KR20130114226A - Human monoclonal antibodies to programmed death 1 (pd-1) and methods for treating cancer using anti-pd-1 antibodies alone or in combination with other immunotherapeutics - Google Patents

Abstract

The present invention provides isolated monoclonal antibodies, specifically human monoclonal antibodies, that specifically bind PD-1 with high affinity. The invention also provides nucleic acid molecules encoding the antibodies of the invention, expression vectors, host cells, and methods of expressing the antibodies of the invention. The invention also provides immunoconjugates, dual specific molecules and pharmaceutical compositions comprising the antibodies of the invention. The present invention also provides methods of detecting PD-1 using anti-PD-1 antibodies and methods of treating various diseases such as cancer and infectious diseases. The present invention also provides a method of treating hyperproliferative diseases, such as cancer, using a combination immunotherapy such as anti-CTLA-4 and anti-PD-1 antibodies in combination. The present invention also provides methods for altering the events of adverse action associated with methods of treating these antibodies independently.

Description

Human monoclonal antibody against predisposed death factor 1 (PD-1), and anti-PD-1 antibody alone or in combination with other immunotherapy agents for cancer treatment {HUMAN MONOCLONAL ANTIBODIES TO PROGRAMMED DEATH 1 (PD-1) ) AND METHODS FOR TREATING CANCER USING ANTI-PD-1 ANTIBODIES ALONE OR IN COMBINATION WITH OTHER IMMUNOTHERAPEUTICS}

The present invention relates generally to the treatment of diseases in humans and to immunotherapy performed in altering the events of the opposite action that occur therein. More specifically, the present invention uses anti-PD-1 antibodies, anti-CTLA-4 and anti-CTLA-4, to alleviate the occurrence or spread of events of opposite action associated with cancer treatment and/or therapy using antibodies, respectively. It relates to performing parallel immunotherapy, such as using an anti-PD-1 antibody in combination.

Protein programmed killing factor 1 (PD-1) is a member of the inhibitory factors belonging to the CD28 group of receptors (including CD28, CTLA-4, ICOS and BTLA). PD-1 is expressed on activated B cells, T cells and bone marrow cells [Agata et al., homologous; Okazaki et al. (2002) Curr. Opin. Immunol. 14: 391779-82; Bennett et al. (2003) J Immunol 170:711-8]. Among the members of this group, CD28 and ICOS were first discovered through the functional effect of increasing the proliferation of T cells by adding a monoclonal antibody [Hutloff et al. (1999) Nature 397:263-266; Hansen et al. (1980) Immunogenics 10:247-260]. PD-1 was discovered through screening for differential expression in apoptotic cells [Ishida et al. (1992) EMBO J 11:3887-95]. Other members of this group, namely CTLA-4 and BTLA, were discovered through screening for differential expression phenomena in cytotoxic T lymphocytes and TH1 cells, respectively. CD28, ICOS and CTLA-4 all have unpaired cysteine residues that allow for homodimerization. In contrast, PD-1 is said to exist as a monomer and does not have a hole cysteine residue that characterizes other members of the CD28 group.

The PD-1 gene is a 55 kDa type I transmembrane protein, which forms part of the Ig phase gene [Agata et al. (1996) Int. Immunol 8:765-72]. PD-1 contains a member of the adjacent immune receptor tyrosine suppression motif (ITIM) and a member of the distal tyrosine-based switch motif (ITSM) [Thomas, M.L. (1995) J Exp Med 181: 1953-6; Vivier, E and Daeron, M (1997) Immunol Today 18:286-91]. PD-1 is structurally similar to CTLA-4, but lacks the MYPPPY motif, which plays an important role in the binding of B7-1 and B7-2. Two ligands for PD-1, PD-L1 and PD-L2, were identified, which are thought to down-regulate the activation of T cells when binding to PD-1 [Freeman et al. (2000) J Exp Med 192: 1027-34; Latchman et al. (2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur J Immunol 32:634-43]. PD-L1 and PD-L2 are both homologs of B7, binding to PD-1 but not other members of the CD28 family. One ligand for PD-1, PD-L1, is found in large quantities in a number of human cancers [Dong et al. (2002) Nat. Med 8:787-9]. Due to the interaction between PD-1 and PD-L1, tumor-invasive lymphocytes decrease, T cell receptor-mediated proliferation decreases, and immune evasion by cancer cells occurs [Dong et al. (2003) J. Mol . Med. 81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100]. Immunosuppression can be reversed by inhibiting the local interaction between PD-1 and PD-L1, and the effect is doubled when the interaction between PD-1 and PD-L2 is blocked [Iwai et al. (2002). Proc. Nat'l Acad. Sci. USA 99: 12293-7; Brown et al. (2003) J. Immunol. 170:1257-66].

PD-1 is a member of the inhibitory factor of the CD28 group expressed on activated B cells, T cells and bone marrow cells [Agata et al., Hom; Okazaki et al. (2002) Curr Opin Immunol 14: 391779-82; Bennett et al. (2003) J Immunol 170:711-8]. In PD-1 deficient animals, various autoimmune phenotypes, such as autoimmune myocardial infarction and lupus-like syndrome with arthritis and nephritis, are induced [Nishimura et al. (1999) Immunity 11: 141-51; Nishimura et al. (2001) Science 291:319-22]. In addition, PD-1 has been shown to play an important role in autoimmune cerebral myelitis, systemic lupus erythematosus, graft-versus-host liver disease (GVHD), type I diabetes, and rheumatoid arthritis [Salama]. Et al. (2003) J Exp Med 198:71-78: Prokunina and Alarcon-Riquelme (2004) Hum Mol Genet 13:R143; Nielsen et al. (2004) Lupus 11:510]. In murine B-cell tumor lines, ITSM of PD-1 ^{was found to be essential to block BCR-mediated Ca 2+} -influx and tyrosine phosphorylation of downstream effector molecules [Okazaki et al. (2001) PNAS 98: 13866- 71].

Therefore, there is a need for an agent that recognizes PD-1 and a method of using such an agent.

Summary of the invention

The present invention provides an isolated monoclonal antibody, specifically a human monoclonal antibody, which binds to PD-1 and exhibits a number of desirable properties. Examples of such properties include high affinity for human PD-1 and substantially no cross-linking reaction with any of human CD28, CTLA-4 or ICOS. In addition, it is believed that the antibodies of the present invention modulate the immune response. Therefore, another aspect of the invention relates to a method of modulating an immune response using an anti-PD-1 antibody. In particular, the present invention is to provide a method of inhibiting the growth of tumor cells in vivo using an anti-PD-1 antibody.

In one aspect, the invention relates to an isolated monoclonal antibody, or antigen-binding portion thereof, wherein the antibody exhibits one or more of the following properties:

(a) the characteristic of binding to human PD-1 and K _D 1×10 ^{-7 M or less;}

(b) a property that does not substantially bind human CD28, CTLA-4 or ICOS;

(c) the property of increasing T cell proliferation in the Mixed Lymphocyte Reaction (MLR) assay;

(d) the property of increasing the production of interferon-gamma in the MLR assay;

(e) the property of increasing the secretion of IL-2 in the MLR assay;

(f) the properties of binding to human PD-1 and cynomolgus monkey PD-1;

(g) a property of inhibiting the binding of PD-L1 and/or PD-L2 to PD-1;

(h) properties that stimulate antigen-specific memory responses;

(i) properties that stimulate an antibody response; And

(j) Properties that inhibit the growth of tumor cells in vivo.

Preferably, the antibody is a human antibody, and in an alternative embodiment the antibody may be, for example, a murine antibody, a chimeric antibody or a humanized antibody.

In a more preferred embodiment, the antibody _{binds to human PD-1 and K D} 5×10 ⁻⁸ M or less, or binds human PD-1 and K _D 1×10 ⁻⁸ M or less, or human PD-1 And K _D 5×10 ^-9 M or less, or human PD-1 and K _D 1×10 ^-8 M and 1×10 ^-10 M.

In another embodiment, the present invention provides an isolated monoclonal antibody or antigen-binding portion thereof, wherein the antibody competes for binding to PD-1 with a reference antibody comprising the following portions: :

(a) a human heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6 and 7; And

(b) a human light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13 and 14.

In various embodiments, the reference antibody comprises the following parts, i.e.

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1; And

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8

or,

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; And

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9

or,

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3; And

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10

or,

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; And

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11

or,

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; And

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12

or,

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6; And

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 13

or,

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7; And

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14

Includes.

In another aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding portion thereof, which _{is a product of or derived from the human V H} 3-33 gene, comprising a heavy chain variable portion, wherein the antibody is PD- Specifically binds to 1. The present invention also provides an isolated monoclonal antibody or antigen-binding portion thereof, which _{is a product of or derived from the human V H} 4-39 gene, comprising a heavy chain variable portion, which antibody is specific for PD-1. Combine with The present invention also provides an isolated monoclonal antibody or antigen-binding portion thereof, which _{is a product of or derived from the human V κ} L6 gene, comprising a light chain variable portion, which antibody specifically binds to PD-1 do. The present invention also provides an isolated monoclonal antibody or antigen-binding portion thereof, which _{is a product of or derived from the human V κ} L15 gene, comprising a light chain variable portion, which antibody specifically binds to PD-1 do.

In a preferred embodiment, the invention provides an isolated monoclonal antibody or antigen-binding portion thereof, comprising the following parts:

(a) the heavy chain variable region of the _{human V H 3-33 gene;} And

(b) light chain variable region of _{human V κ L6 gene}

[Here, the antibody specifically binds to PD-1].

In other preferred embodiments, the invention provides an isolated monoclonal antibody or antigen-binding portion thereof, comprising the following parts:

(a) the heavy chain variable region of the _{human V H 4-39 gene;} And

(b) light chain variable region of _{human V κ L15 gene}

[Here, the antibody specifically binds to PD-1].

In another aspect, the present invention provides an isolated monoclonal antibody or antigen-binding portion thereof, comprising the following parts:

A heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences; And

Light chain variable region comprising CDR1, CDR2 and CDR3 sequences

[here,

(a) the heavy chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34 and 35 and conservative variants thereof;

(b) the light chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 51, 52, 53, 54, 55 and 56 and conservative variants thereof;

(c) The antibody specifically binds to human PD-1.

Preferably, the heavy chain variable region CDR2 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 22, 23, 24, 25, 26, 27 and 28 and conservative variants thereof; The light chain variable region CDR2 sequence includes an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 43, 44, 45, 46, 47, 48 and 49 and conservative variants thereof. Preferably, the heavy chain variable region CDR1 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 15, 16, 17, 18, 19, 20 and 21 and conservative variants thereof; The light chain variable region CDR1 sequence includes an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 36, 37, 38, 39, 40, 41 and 42 and conservative variants thereof.

In another aspect, the present invention provides an isolated monoclonal antibody or antigen-binding portion thereof comprising a heavy chain variable region and a light chain variable region, wherein

(a) the heavy chain variable portion comprises an amino acid sequence that is 80% or more homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6 and 7;

(b) the light chain variable region comprises an amino acid sequence that is 80% or more homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13 and 14;

(c) the antibody binds to human PD-1 with a K _D 1×10 ^-7 M or less;

(d) The antibody does not substantially bind human CD28, CTLA-4 or ICOS.

In a preferred embodiment, the antibody further comprises one or more of the following properties:

(a) the property of increasing the proliferation of T cells in the MLR assay;

(b) the property of increasing the production of interferon-gamma in the MLR assay; or

(c) Characteristics of increasing IL-2 secretion in the MLR assay.

Additionally or alternatively, the antibody may comprise one or more of the other properties listed above.

In a preferred embodiment, the invention provides an isolated monoclonal antibody or antigen-binding portion thereof, comprising the following parts:

(a) a heavy chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20 and 21;

(b) a heavy chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 23, 24, 25, 26, 27 and 28;

(c) a heavy chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34 and 35;

(d) a light chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 37, 38, 39, 40, 41 and 42;

(e) a light chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 43, 44, 45, 46, 47, 48 and 49;

(f) a light chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 51, 52, 53, 54, 55 and 56

[Here, the antibody specifically binds to PD-1].

Preferred combinations include:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 15;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 22;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 29;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 36;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 43; And

(f) a light chain variable region CDR3 comprising SEQ ID NO: 50.

Other preferred combinations include:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 16;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 23;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 30;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 37;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 44; And

(f) a light chain variable region CDR3 comprising SEQ ID NO: 51.

Other preferred combinations include:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 17;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 24;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 31;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 38;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 45; And

(f) a light chain variable region CDR3 comprising SEQ ID NO: 52.

Other preferred combinations include:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 18;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 25;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 32;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 39;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 46; And

(f) a light chain variable region CDR3 comprising SEQ ID NO: 53.

Other preferred combinations include:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 19;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 26;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 33;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 40;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 47; And

(f) a light chain variable region CDR3 comprising SEQ ID NO: 54.

Other preferred combinations include:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 20;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 27;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 34;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 41;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 48; And

(f) a light chain variable region CDR3 comprising SEQ ID NO: 55.

Other preferred combinations include:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 21;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 28;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 35;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 42;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 49; And

(f) a light chain variable region CDR3 comprising SEQ ID NO: 56.

Other preferred antibodies or antigen-binding portions thereof of the invention comprise the following parts:

(a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6 and 7; And

(b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13 and 14

[Here, the antibody specifically binds to PD-1].

Preferred combinations include:

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1; And

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.

Other preferred combinations include the following.

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; And

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9.

Other preferred combinations include the following.

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3; And

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10.

Other preferred combinations include the following.

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; And

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11.

Other preferred combinations include the following.

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; And

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12.

Other preferred combinations include the following.

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6; And

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 13.

Other preferred combinations include the following.

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7; And

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14.

The antibody of the present invention may be, for example, a full-length antibody such as an IgG1 or IgG4 same reference sample type. Alternatively, the antibody of the invention may be an antibody fragment such as a Fab or Fab'2 fragment or a single chain antibody.

The present invention also provides an immunoconjugate in which the antibody or antigen-binding portion thereof of the present invention is bound to a therapeutic agent such as a cytotoxin or a radioactive isotope. The present invention also provides a bispecific molecule in which the antibody or antigen-binding portion thereof of the present invention is bound to a second functional portion having a different binding specificity than the antibody or antigen-binding portion thereof.

A composition comprising the antibody or antigen-binding portion thereof of the present invention, or an immunoconjugate or dual specific molecule, and a pharmaceutically acceptable carrier is also provided.

Nucleic acid molecules encoding the antibody or antigen-binding portion thereof of the present invention, and expression vectors containing such nucleic acids, and host cells containing such expression vectors are also included in the present invention. Furthermore, the present invention provides a transgenic mouse comprising human immunoglobulin heavy and light chain transgenes, wherein the mouse expresses the antibody of the present invention, and the hybridoma produced from the mouse is the present invention. Produce the antibody of the invention.

In another aspect, the present invention provides a method of modulating an immune response in a subject, the method comprising administering to the subject the antibody or antigen-binding portion thereof of the present invention, thereby modulating the immune response in the subject. do. Preferably, the antibody of the present invention enhances, promotes or increases an immune response in a subject.

In a further aspect, the invention provides a method of inhibiting the growth of tumor cells in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding portion thereof. . Although other anti-PD-1 antibodies may be used instead (or may be used in conjunction with the anti-PD-1 antibodies of the present invention), it is preferred to use the antibodies of the present invention in the above method. For example, anti-PD-1 antibodies that are chimeric, humanized or fully human can be used in methods of inhibiting the growth of tumors.

In a further aspect, the invention provides a method of treating an infectious disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding portion thereof. Although other anti-PD-1 antibodies may be used instead (or may be used in conjunction with the anti-PD-1 antibodies of the present invention), it is preferred to use the antibodies of the present invention in the above method. For example, anti-PD-1 antibodies that are chimeric, humanized or completely human can be used in methods of treating infectious diseases.

In addition, the present invention provides a method for enhancing an immune response to an antigen in a subject, the method comprising: (i) an antigen; And (ii) administering the anti-PD-1 antibody or antigen-binding portion thereof to the subject, thereby enhancing an immune response to the antigen in the subject. The antigen can be, for example, a tumor antigen, a viral antigen, a bacterial antigen or an antigen derived from a pathogen. Although other anti-PD-1 antibodies can be used instead (or can be used in conjunction with the anti-PD-1 antibodies of the invention), it is preferred to use the antibodies of the invention in the above method. For example, anti-PD-1 antibodies that are chimeric, humanized or wholly human can be used in a method of enhancing an immune response to an antigen in an individual.

The invention also provides a method of making a "second generation" anti-PD-1 antibody based on the sequence of the anti-PD-1 antibody provided herein. For example, the present invention provides a method of making an anti-PD-1 antibody, comprising the following steps:

(a) (i) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20 and 21; And/or a CDR2 sequence selected from the group consisting of SEQ ID NOs: 22, 23, 24, 25, 26, 27 and 28; And/or a heavy chain variable region antibody sequence comprising a CDR3 sequence selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34 and 35; Or (ii) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 36, 37, 38, 39, 40, 41 and 42; And/or a CDR2 sequence selected from the group consisting of SEQ ID NOs: 43, 44, 45, 46, 47, 48 and 49; And/or providing a light chain variable region antibody sequence comprising a CDR3 sequence selected from the group consisting of SEQ ID NOs: 50, 51, 52, 53, 54, 55 and 56;

(b) modifying one or more amino acid residues present in one or more variable region antibody sequences to generate one or more modified antibody sequences, wherein the variable region antibody sequence is a heavy chain variable region antibody sequence and a light chain variable region antibody sequence. Is selected from; And

(c) expressing the modified antibody sequence as a protein.

Other features and advantages related to the present invention will become more apparent through the following detailed description and examples (however, they should not be construed as limiting the present invention). All references, GenBank registrations, patents and published patent applications cited throughout this application are incorporated herein by reference.

Best way to carry out the present invention

In one aspect, the invention relates to an isolated monoclonal antibody that specifically binds to PD-1, in particular to a human monoclonal antibody. In certain embodiments, the antibody of the invention has one or more desirable functional properties, such as high affinity binding properties for PD-1, lack of cross-reactivity with other CD28 family members, the ability to promote T cell proliferation. , IFN-γ and/or IL-2 secretion properties in mixed lymphocyte responses, binding inhibition properties with one or more PD-1 ligands (e.g., PD-L1 and/or PD-L2), cynomolgus monkey PD- It shows the ability to cross-link with 1, the ability to promote an antigen-specific memory response, the ability to promote an antibody response, and/or the ability to inhibit the growth of tumor cells in vivo. Additionally or alternatively, the antibodies of the invention specifically derive from the germline sequences of the heavy and light chains and/or comprise a CDR region comprising certain structural features, such as certain amino acid sequences. In another aspect, the present invention relates to the use of a monoclonal antibody that specifically binds to PD-1 and a monoclonal antibody that specifically binds to CTLA-4.

The present invention includes, for example, an isolated antibody of the present invention, a method of making such an antibody, an immunoconjugate and a bispecific molecule comprising such an antibody, and an antibody, immunoconjugate or bispecific molecule of the present invention. Provides a pharmaceutical composition.

In another aspect, the present invention relates to a method of inhibiting the growth of tumor cells in a subject using an anti-PD-1 antibody. As described herein, anti-PD-1 antibodies can inhibit the growth of tumor cells in vivo. The present invention also modifies the immune response, treats a disease such as cancer or infectious disease, or promotes a protective autoimmune response, or (e.g., by administering an antigen of interest and anti-PD-1 together) It also relates to methods of using antibodies to promote antigen-specific immune responses.

In order to more easily understand the present invention, first, some terms have been defined. Additional definitions are presented throughout the detailed description.

"Programmed Death 1", "Programmed Cell Death 1", "Protein PD-1", "PD-1", "PD1", "PDCD1", "hPD-1 "And "hPD-I" are used interchangeably and include variants, subtypes, homologous species of human PD-1, and analogs having one or more epitopes in common with PD-1. Complete PD-1 sequence Is registered under GenBank Approval No. U64863.

"Cytotoxic T lymphocyte-associated antigen-4," "CTLA-4," "CTLA4," "CTLA-4 antigen" and "CD152" (eg, Murata, Am . J. Pathol. (1999) 155:453-460] are used interchangeably, including variants, subtypes, homologous species of human CTLA-4, and analogs having one or more epitopes common to CTLA-4 [eg , Balzano (1992) Int. J. Cancer Suppl. 7:28-32]. The complete CTLA-4 nucleic acid sequence is registered under GenBank Accession No. L15006.

The term "immune response" refers to a pathogen-infiltrating body, a pathogen-infected cell or tissue, a cancer cell, or (in the case of an autoimmune response or pathogenic inflammation), selectively damaging or damaging normal human cells or tissues. It refers to the action of the lymphocytes, antigen-providing cells, phagocytic cells, granulocytes, and soluble macromolecules (eg, antibodies, cytokines and complement) produced by the cells or liver to destroy or eliminate.

By “signal transduction pathway” is meant a biochemical relationship between various signal transduction molecules that serve to transfer signals from one site of a cell to another. As used herein, the phrase “cell surface receptor” includes molecules and complexes of molecules that are capable of, for example, receiving a signal and transporting the received signal across the plasma membrane of a cell. An example of the "cell surface receptor" of the present invention is the PD-1 receptor.

As used herein, the term “antibody” includes whole antibodies and any antigen-binding fragments thereof (ie, “antigen-binding portions”) or single chains. "Antibody" refers to a glycoprotein or antigen-binding portion thereof in which two or more heavy chains (H) and two or more light chains (L) are linked to each other by a disulfide bond. Each heavy chain consists of a heavy chain variable region ( _{abbreviated herein as V H} ) and a heavy chain constant region. The heavy chain constant region consists of three domains, C _H1 , C _H2 and CH ₃ . Each light chain consists of a light chain variable region ( _{abbreviated herein as V L} ) and a light chain constant region. The light chain constant region consists of one domain, that is, C _L. The V _H and V _L regions may also be subdivided into hypermutation regions, that is, a more conservative region, that is, a complementarity determining region (CDR) in which a frame region (FR) is inserted in the middle. Each of V _H and V _L consists of 3 CDRs and 4 FRs in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody may mediate the binding of an immunoglobulin to a host's tissues or factors, such as the various cells of the immune system (e.g., effector cells) and the first component (C1q) of the traditional complement system.

The term "antigen-binding portion" (or simply "antigen portion") of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (eg, PD-1). Is to mean. It has been found that the antigen-binding function of an antibody can be performed by fragments of full-length antibodies. Examples of binding fragments belonging to the term "antigen-binding portion" of an antibody include (i) Fab fragments, ie, monovalent fragments consisting of _{V L} , V _H , C _L and C _{H1 domains;} (ii) F(ab') ₂ fragment, that is, a bivalent fragment consisting of two Fab fragments bound by disulfide bonds at the hinge site; (iii) an Fd fragment consisting of the _{V H} and C _{H1 domains;} (iv) an Fv fragment consisting _{of the V L} and V _H domains of a single arm of the antibody; (v) _{a dAb fragment consisting of the V H} domain [Ward et al., (1989) Nature 341 :544-546]; And (vi) an isolated complementarity determining site (CDR). In addition, even though the two domains of the Fv fragment, V _L and V _H , are encoded by individual genes, these domains are known as monovalent molecules (single-chain Fv (scFv) using a recombination method). Yes; see, for example, Bird et al. (1988) Science 242 :423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85 :5879-5883) to form V _L and V _H It can be linked through a synthetic linker that allows the sites to be produced as paired single protein chains. Such single chain antibodies are also included in the term "antigen-binding portion" of an antibody. Such antibody fragments are obtained using conventional techniques known to the person skilled in the art, and these fragments are screened for utility in the same manner as in the case of antibodies in their original state.

As used herein, "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities [for example, as an isolated antibody that specifically binds to PD-1, PD-1 It does not contain an antibody that specifically binds to other antigens]. However, isolated antibodies that specifically bind to PD-1 may have cross-reactivity against other antigens, such as PD-1 molecules derived from other species. In addition, the isolated antibody may be substantially free of other cellular and/or chemical substances.

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules in a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The term "human antibody" as used herein is meant to include antibodies having variable regions derived from human germline immunoglobulin sequences, both the framework region and the CDR region. In addition, if the antibody contains a constant region, this constant region is also derived from the human germline immunoglobulin sequence. Human antibodies of the present invention contain amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). Can include. However, the term “human antibody” as used herein is not meant to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as mice, have been grafted into human framework sequences.

The term "human monoclonal antibody" refers to an antibody that has a variable region derived from a human germline immunoglobulin sequence and exhibits a single binding specificity, both of the frame region and the CDR region. In one embodiment, the human monoclonal antibody is obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome in which a human heavy chain transgene and a light chain transgene are fused to an immortalized cell. It is produced by hybridomas containing cells.

As used herein, the term "recombinant human antibody" refers to any human antibody produced, expressed, produced or isolated by a recombinant method, such as (a) a human immunoglobulin gene or a hybridoma produced therefrom (hereinafter For example, transgenic or transchromosomal, an antibody isolated from an animal (e.g., mouse), (b) a host cell transformed to express a human antibody, e.g., trans An antibody isolated from a transfectoma, (c) an antibody isolated from a recombinant, combinatorial human antibody library, and (d) any other method comprising splicing a human immunoglobulin gene sequence to other DNA sequences. It includes an antibody produced, expressed, produced or isolated by. Such recombinant human antibodies have variable regions whose framework regions and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, in such a recombinant human antibody, an in vitro mutagenesis method (or a somatic cell mutagenesis method in vivo when an animal that is transgenic for human Ig sequence is used) may proceed. _{The amino acid sequence of the V H} and V _L regions of the antibody is a sequence derived from and related to the human germ line V _H and V _L , and is a sequence that cannot exist in the human antibody germ line repertoire in nature.

As used herein, the "same reference sample" means a group of antibodies (eg, IgM or IgG1) encoded by a heavy chain constant region gene.

The phrases "antibody recognizing an antigen" and "antibody specific for an antigen" are used interchangeably herein with "antibody that specifically binds to an antigen".

The term “human antibody derivative” is intended to mean any modified form of a human antibody, eg, an antibody and other agents or conjugates of an antibody.

The term "humanized antibody" refers to an antibody in which a CDR sequence derived from the germline of another mammalian species, such as a mouse, has been grafted onto a human framework sequence. Additional framework region modifications can be made within the human framework sequence.

The term "chimeric antibody" means that the variable region sequence is derived from one species and the constant region sequence is derived from another species. For example, the variable region sequence is derived from a mouse antibody, and the constant region sequence is a human antibody. It means an antibody derived from.

As used herein, the "specifically binding to human PD-1" antibody binds to human PD-1 and K _D 1×10 ^-7 M or less, more preferably K _D 5×10 ^-8 M or less It refers to an antibody that binds to, more preferably K _D 1 × 10 ^-8 M or less, or more preferably K _D 5 × 10 ^{-9 M or less.}

The term "K _assoc " or "K _a ", as used herein, refers to the binding rate of a particular antibody-antigen interaction, while the terms _{"K dis} " or "K _{d" as used herein are} , Refers to the rate of dissociation of a specific antibody-antigen interaction. In the present application, the _{term "K D} " refers to a dissociation constant, and _{the ratio of K d} :K _a (ie, K _d /K _a ) is expressed as a molar concentration (M). _{K D} values for antibodies can be determined using methods well established in the art. A preferred method for measuring the K _D value of an antibody is the surface plasmon resonance method, preferably using a biosensor system, such as the Biacore® system.

As used herein, the term "high affinity" for an IgG antibody means that the K _D value of the antibody for the target antigen is 10 ^-8 M or less, more preferably 10 ^-9 M or less and even more preferably It means the case of ^{10 -10 M or less.} However, "high affinity" binding may vary for different antibody same reference samples. For example, "high affinity" binding to an IgM same reference sample means that the antibody has a K _D value of 10 ^-7 M or less, more preferably 10 ^-8 M or less, even more preferably 10 ^-9 M or less. Is to mean.

The term “treatment” or “treatment” refers to a condition (e.g., a disease), a condition that heals, cures, alleviates, alleviates, alters, corrects, or improves the symptoms of the condition To improve or affect, or to prevent or delay the occurrence of symptoms, complications, or biochemical signs of such a disease, or to delay or inhibit further progression of a disease, condition, or disease, statistics It refers to the process of administering the active agent in a scientifically significant manner.

As used herein, “adverse event” (AE) refers to an undesirable, generally unintended, or even undesirable indication (eg, finding an abnormality in the laboratory), symptom, or disease associated with the conduct of a medical treatment. do. For example, an adverse event may be associated with activation of the immune system following treatment or proliferation of immune system cells (eg, T cells). Medical treatment may result in more than one related AE, and each AE may be the same or different in severity. By "adverse event changeable" method is meant a treatment modality that reduces the incidence and/or severity of one or more AEs resulting from the application of different treatment modalities.

As used herein, "hyperproliferative disease" refers to a condition in which cell growth increases above normal levels. For example, hyperproliferative diseases or diseases include malignant diseases (e.g., esophageal cancer, colon cancer, bile duct cancer) and non-malignant diseases (e.g., atherosclerosis, benign hyperplasia, benign prostatic hyperplasia). .

As used herein, "subtherapeutic dose" refers to a dosage of a therapeutic compound (eg, an antibody), and is used solely for the treatment of hyperproliferative diseases (eg, cancer). When administered as, it means an amount less than a general dosage or a conventional dosage of the therapeutic compound. For example, a dose below the therapeutic dose of CTLA-4 antibody is the amount when this antibody is administered once (less than about 3 mg/kg), that is, an amount that is less than the known dose of anti-CTLA-4 antibody. .

It is to be understood that the use of an alternative (eg, “or”) is meant to mean one, two, or any combination of these alternatives. As used herein, the indefinite article “a” or “an” is to be understood to mean “one or more” of any of the ingredients mentioned or listed.

As used herein, "about" or "essentially including" means to be within a tolerance for a specific value determined by the person skilled in the art, in part, that is, how the value was measured or determined. However, it will depend on how far the limits of the measurement system are. For example, "about" or "essentially including" may mean that it falls within one or more standard deviations per practice in the art. Alternatively, "about" or "essentially including" may mean a range of up to 20%. In addition, particularly with respect to a biological system or process, the terms may mean a certain level of quantity or up to 5 times its value. When a specific value is given in the present application and in the claims, unless stated otherwise, "about" or "essentially including" is meant to assume that it is within the tolerance for such specific value.

As described herein, any concentration range,% range, ratio range, or integer range, unless stated otherwise, includes any integer value falling within the range recited herein, and where appropriate, fractions thereof (e.g. For example, it will be understood to include tenths of an integer and one hundredths).

As used herein, the term “individual” is meant to include any human or non-human animal. The term "non-human animal" refers to all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. It is meant to include. Except where otherwise noted, “patient” or “subject” are used interchangeably.

Various aspects of the invention are described in more detail in the following subsections.

Anti-PD-1 antibody

The antibody of the present invention is characterized by having a functional characteristic or characteristic of the antibody alone. For example, this antibody specifically binds to PD-1 [for example, it binds to human PD-1 and is capable of cross-reacting with PD-1 from other species such as cynomolgus monkeys. I can]. Preferably, the antibody of the present invention binds PD-1 with high affinity, eg, K _D 1×10 ⁻⁷ M or less. Anti-PD-1 antibodies of the invention preferably exhibit one or more of the following characteristics:

(a) the characteristic of binding to human PD-1 and K _D 1×10 ^{-7 M or less;}

(b) a property that does not substantially bind human CD28, CTLA-4 or ICOS;

(c) the property of increasing T cell proliferation in a mixed lymphocyte response (MLR) assay;

(d) the property of increasing the production of interferon-gamma in the MLR assay;

(e) the property of increasing the secretion of IL-2 in the MLR assay;

(f) the properties of binding to human PD-1 and cynomolgus monkey PD-1;

(g) a property of inhibiting the binding of PD-L1 and/or PD-L2 to PD-1;

(h) properties that promote antigen-specific memory responses;

(i) properties that promote antibody response; And

(j) Properties that inhibit the growth of tumor cells in vivo.

Preferably, the antibody binds to human PD-1 and K _D 5×10 ⁻⁸ M or less, or binds human PD-1 and K _D 1×10 ⁻⁸ M or less, or human PD-1 and K _D 5 It binds to less than × 10 ^-9 M, or binds human PD-1 with K _D 1 × 10 ^-8 M and 1 × 10 ^-10 M.

The antibody of the present invention can be expressed by combining any number of features (eg, two, three, four, or five or more features) among the features listed above.

Standard assays for evaluating the binding ability of an antibody to PD-1 are known in the art, examples of which include ELISA, Western Blot and RIA. The binding kinetics (eg, binding affinity) of an antibody can also be assessed by standard assays known in the art, such as Biacore assays. A test method suitable for evaluating any of the features listed above is described in detail in the Examples below.

Monoclonal antibodies 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4

Preferred antibodies of the invention are the human monoclonal antibodies 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 [isolated and structurally characterized as described in Examples 1 and 2]. _{The V H} amino acid sequences of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6 and 7, respectively. _{The V L} amino acid sequences of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shown in SEQ ID NOs: 8, 9, 10, 11, 12, 13 and 14, respectively.

If each of these antibodies is capable of binding to PD-1, the V _H and V _L sequences can be "mixed and matched" to form other anti-PD-1 binding molecules of the invention. Binding of such “mixed and matched” antibodies to PD-1 can be tested using a binding assay (eg, ELISA) as described above and in the Examples. Preferably, when the V _H and V _L chains are mixed and matched, _{a V H} sequence derived from a _{particular V H} /V _L pair is substituted with a structurally similar V _{H sequence.} Similarly, it is preferred that _{a V L} sequence derived from a _{particular V H} /V _L pair is substituted with a structurally similar V _{L sequence.}

Therefore, in one aspect, the present invention provides an isolated monoclonal antibody or antigen-binding portion thereof, comprising the following parts:

(a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6 and 7; And

(b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13 and 14

[Here, the antibody specifically binds to PD-1, preferably human PD-1].

Preferred combinations of heavy and light chains include the following:

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1; And

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8

or,

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; And

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9

or,

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3; And

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10

or,

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; And

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11

or,

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; And

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12

or,

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6; And

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 13

or,

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7; And

(b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14.

In another embodiment, the invention provides an antibody comprising heavy and light chain CDR1, CDR2 and CDR3 of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4, or a combination thereof. _{The amino acid sequences of the V H} CDR1s of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shown in SEQ ID NOs: 15, 16, 17, 18, 19, 20 and 21, respectively. _{The amino acid sequences of the V H} CDRs of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shown in SEQ ID NOs: 22, 23, 24, 25, 26, 27 and 28, respectively. _{The amino acid sequences of the V H} CDR3 of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shown in SEQ ID NOs: 29, 30, 31, 32, 33, 34 and 35, respectively. _{The amino acid sequences of V κ} CDR1 of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shown in SEQ ID NOs: 36, 37, 38, 39, 40, 41 and 42, respectively. _{The amino acid sequences of the V κ} CDR2 of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shown in SEQ ID NOs: 43, 44, 45, 46, 47, 48 and 49, respectively. _{The amino acid sequences of the V κ} CDR3 of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shown in SEQ ID NOs: 50, 51, 52, 53, 54, 55 and 56, respectively. CDR regions were indicated according to the Kabat system [Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242].

If the antibodies can each bind to PD-1 and antigen-binding specificity is primarily provided by the CDR1, CDR2 and CDR3 regions, the V _H CDR1, CDR2 and CDR3 sequences and the V _κ CDR1, CDR2 and CDR3 sequences Is "mixed and matched" (ie, although each antibody must contain V _H CDR1, CDR2 and CDR3 and V _κ CDR1, CDR2 and CDR3, CDRs derived from different antibodies can be mixed and matched. ], it is possible to form the RLXK anti-PD-1 binding molecule of the present invention. Whether such “mixed and matched” antibodies bind to PD-1 can be tested using a binding assay (eg, ELISA, Biacore assay) as described above and described in the Examples. Preferably, when V _H CDR sequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequences derived from a _{particular V H sequence are replaced with structurally similar CDR sequence(s).} Similarly, when V _κ CDR sequences are mixed and matched, it is preferred that the CDR1, CDR2 and/or CDR3 sequences derived from a _{particular V κ sequence are replaced with structurally similar CDR sequence(s).} The novel V _H and V _L sequences are structurally similar to one or more V _H and/or V _L CDR region sequences (as disclosed herein as for monoclonal antibodies 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4. It will be appreciated by those skilled in the art that it can be generated by substitution with a sequence derived from a CDR sequence).

Therefore, in another aspect, the present invention provides an isolated monoclonal antibody or antigen-binding portion thereof, comprising the following parts:

(a) a heavy chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20 and 21;

(b) a heavy chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 23, 24, 25, 26, 27 and 28;

(c) a heavy chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34 and 35;

(d) a light chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 37, 38, 39, 40, 41 and 42;

(e) a light chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 43, 44, 45, 46, 47, 48 and 49;

(f) a light chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 51, 52, 53, 54, 55 and 56

[Here, the antibody specifically binds to PD-1, preferably human PD-1].

In a preferred embodiment, the antibody comprises the following moieties:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 15;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 22;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 29;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 36;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 43; And

(f) a light chain variable region CDR3 comprising SEQ ID NO: 50.

In a preferred embodiment, the antibody comprises the following moieties:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 16;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 23;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 30;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 37;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 44; And

(f) a light chain variable region CDR3 comprising SEQ ID NO: 51.

In a preferred embodiment, the antibody comprises the following moieties:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 17;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 24;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 31;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 38;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 45; And

(f) a light chain variable region CDR3 comprising SEQ ID NO: 52.

In a preferred embodiment, the antibody comprises the following moieties:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 18;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 25;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 32;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 39;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 46; And

(f) a light chain variable region CDR3 comprising SEQ ID NO: 53.

In other preferred embodiments, the antibody comprises the following moieties:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 19;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 26;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 33;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 40;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 47; And

(f) a light chain variable region CDR3 comprising SEQ ID NO: 54.

Antibodies with specific germline sequences

In certain embodiments, the antibodies of the invention comprise a heavy chain variable region derived from a specific germline heavy chain immunoglobulin gene and/or a light chain variable region derived from a specific germline light chain immunoglobulin gene.

For example, in a preferred embodiment, the present invention provides an isolated monoclonal antibody or antigen-binding portion thereof, comprising a heavy chain variable region that is a product of or derived from the _{human V H 3-33 gene, wherein} The antibody specifically binds to PD-1, preferably human PD-1. In another preferred embodiment, the present invention provides an isolated monoclonal antibody or antigen-binding portion thereof, comprising a heavy chain variable region derived from or a product _{of the human V H 4-39 gene, wherein the antibody is} It specifically binds to PD-1, preferably human PD-1. In a further other preferred embodiment, the present invention provides an isolated monoclonal antibody or antigen-binding portion thereof, comprising a light chain variable region derived from or a product _{of the human V κ L6 gene, wherein the antibody is} It specifically binds to PD-1, preferably human PD-1. In a further other preferred embodiment, the present invention provides an isolated monoclonal antibody or antigen-binding portion thereof, comprising a light chain variable region derived from or a product _{of the human V κ L15 gene, wherein the antibody is} It specifically binds to PD-1, preferably human PD-1. In yet other preferred embodiments, the invention provides an isolated monoclonal antibody or antigen-binding portion thereof, the antibody comprising:

(a) _{a product of or derived from a human V H} 3-33 or 4-39 gene (ie, a gene encoding an amino acid sequence set forth in SEQ ID NO: 71 or 73, respectively);

(b) _{a light chain variable region which is the product of or derived from a human V κ} L6 or L15 gene (ie, a gene encoding an amino acid sequence set forth in SEQ ID NO: 72 or 74, respectively);

(c) specifically binds to PD-1.

Examples of antibodies having V _H and V _κ of V _H 3-33 and V _κ L6 are 17D8, 2D3, 4H1, 5C4 and 7D3, respectively. Examples of antibodies with V _H and V _κ of V _H 4-39 and V _κ L15 are 4A11 and 5F4, respectively.

As used herein, a human antibody is a heavy or light chain that is a product of a specific germline sequence or is derived therefrom, if the variable portion of the antibody is obtained from a system using a human germline immunoglobulin gene. It includes a variable part. Such systems involve immunizing transgenic mice carrying human immunoglobulin genes with an antigen of interest, or screening a library of human immunoglobulin genes displayed on phage using the antigen of interest. Human antibodies that are "products of" or "derived from" the human germline immunoglobulin sequence compare the amino acid sequence of the human antibody with the amino acid sequence of the human germline immunoglobulin, and are closest to the sequence of the human antibody. It can be identified by selecting a human germline immunoglobulin sequence that has the sequence (ie, has the greatest% sequence identity). Human antibodies that are "products of" or "derived from" certain human germline immunoglobulin sequences are human antibodies, for example due to the deliberate introduction of naturally-occurring somatic mutations or site-oriented mutations, There may be differences in amino acids compared to germline sequences. However, the selected human antibody is typically 90% or more identical in amino acid sequence to the amino acid sequence encoded by the human germline immunoglobulin gene, and the germline immunoglobulin amino acid sequence of other species (e.g. Family sequence), it contains amino acid residues that make the human antibody distinct as human. In any case, the human antibody may be at least 95%, or at least 96%, 97%, 98% or 99% identical in amino acid sequence relative to the amino acid sequence encoded by the germline immunoglobulin gene. Typically human antibodies derived from certain human germline sequences will exhibit amino acid differences in less than 10 amino acids from the amino acid sequence encoded by the human germline immunoglobulin gene. In any case, human antibodies may exhibit amino acid differences in less than 5, or less than 4, 3, 2, or 1 amino acids from the amino acid sequence encoded by the germline immunoglobulin gene.

Homologous antibody

In another embodiment, the antibody of the invention comprises a heavy and light chain variable region comprising an amino acid sequence homologous to the amino acid sequence of a preferred antibody described herein, wherein the antibody is an anti-PD-1 antibody of the invention Retains the desirable functional properties of

For example, the present invention provides an isolated monoclonal antibody or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein:

(a) the heavy chain variable portion comprises an amino acid sequence that is 80% or more homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6 and 7;

(b) the light chain variable portion comprises an amino acid sequence that is 80% or more homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13 and 14;

The antibody has one or more of the following properties:

(c) human PD-1 and K _D 1 × 10 ^-7 M or less binding properties;

(d) a property that does not substantially bind human CD28, CTLA-4 or ICOS;

(e) the property of increasing T cell proliferation in the MLR assay;

(f) the property of increasing the production of interferon-gamma in the MLR assay;

(g) the property of increasing the secretion of IL-2 in the MLR assay;

(h) properties of binding to human PD-1 and cynomolgus monkey PD-1;

(i) a property of inhibiting the binding of PD-L1 and/or PD-L2 to PD-1;

(j) properties that promote antigen-specific memory responses;

(k) a property that promotes an antibody response; And

(l) Properties that inhibit the growth of tumor cells in vivo.

In other embodiments, the V _H and/or V _L amino acid sequence may be 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the sequences presented above. High homology between the V _H and V _L regions of the presented sequences (i.e., 80% or more) V _H, and an antibody having the V _L region is SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, After mutation (e.g., site-oriented or PCR-mediated mutation) of the nucleic acid sequence encoding 64, 65, 66, 67, 68, 69 and 70, using the functional assay described herein, It can be obtained by testing whether or not the encoded modified antibody has acquired a function (ie, the function set forth in (c) to (l) above).

In the present application, the percent homology between the two amino acid sequences is the same as the percent identity between the two sequences. The percent identity between two sequences is a function of the number of identical positions shared by the sequence [i.e.,% homology = (number of identical positions/number of total positions) × 100], for optimal alignment of the two sequences. It is a concept that considers the number of gaps that need to be introduced and the length of each gap. Sequence comparison and percent identity determination between two sequences can be performed using a mathematical algorithm, as described in the non-limiting examples below.

The percent identity between the two amino acid sequences is described in E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), which can be measured using the algorithm, wherein the algorithm utilizes the PAM120 weight residue table, Gap length penalty = 12 and gap penalty = 4. In addition, the% identity between the two amino acid sequences is described in Needleman and Wunsch (J. Mol. Biol. 48:444). -453 (1970)], which utilizes a Blossum 62 matrix or a PAM250 matrix, with gap weight = 16, 14, 12, 10, 8, 6 or 4, length weight = 1, 2, 3, 4, 5, or 6 is used in conjunction with the GAP program of the GCG software package (available at www.gcg.com).

Additionally or alternatively, protein sequences of the invention can also be used as “query sequences” to perform searches against publicly available databases, for example to identify relevant sequences. . This search was carried out by the XBLAST program (version 2.0) [Altschul et al. (1990) J. Mol. Biol. 215:403-10]. BLAST protein search is performed using the XBLAST program (score = 50, character length = 3), and an amino acid sequence homologous to the antibody molecule of the present invention can be obtained. In order to obtain a gapped arrangement for comparison purposes, a Gapped BLAST (Gapped BLAST) program can be used [Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402]. When utilizing BLAST and Gap Formation BLAST programs, the default parameters of each program (eg, XBLAST and NBLAST) can be used (see www.ncbi.nlm.nih.gov).

Antibodies containing conservative modifications

In certain embodiments, the antibody of the invention comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, wherein at least one of the CDR sequences is herein A specific amino acid sequence based on a preferred antibody disclosed in (e.g., 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4) and conservative variants thereof, wherein the antibody is an anti- It retains the desirable functional properties of the PD-1 antibody. Therefore, the present invention provides an isolated monoclonal antibody or antigen-binding portion thereof comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, wherein:

(a) the heavy chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 29, 30, 31, 32, 33, 34 and 35 and conservative variants thereof;

(b) the light chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 50, 51, 52, 53, 54, 55 and 56 and conservative variants thereof;

The antibody has one or more of the following properties:

(c) human PD-1 and K _D 1 × 10 ^-7 M or less binding properties;

(d) a property that does not substantially bind human CD28, CTLA-4 or ICOS;

(e) the property of increasing T cell proliferation in the MLR assay;

(f) the property of increasing the production of interferon-gamma in the MLR assay;

(g) the property of increasing the secretion of IL-2 in the MLR assay;

(h) properties of binding to human PD-1 and cynomolgus monkey PD-1;

(i) a property of inhibiting the binding of PD-L1 and/or PD-L2 to PD-1;

(j) properties that promote antigen-specific memory responses;

(k) a property that promotes an antibody response; And

(l) Properties that inhibit the growth of tumor cells in vivo.

In a preferred embodiment, the heavy chain variable region CDR2 sequence of the invention comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 22, 23, 24, 25, 26, 27 and 28 and conservative variants thereof; The light chain variable region CDR2 sequence includes an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 43, 44, 45, 46, 47, 48 and 49 and conservative variants thereof. In a preferred embodiment, the heavy chain variable region CDR1 sequence of the invention comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 15, 16, 17, 18, 19, 20 and 21 and conservative variants thereof; The light chain variable region CDR1 sequence includes an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 36, 37, 38, 39, 40, 41 and 42 and conservative variants thereof.

As used herein, the term “conservative sequence modification” is intended to mean an amino acid modification that has little or no effect on the binding properties of an antibody containing an amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-oriented mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are when one amino acid residue is substituted with another amino acid residue having a similar side chain. A group of amino acid residues having similar side chains is defined in the art. Such groups include amino acids having basic side chains (eg, lysine, arginine, and histidine), amino acids having acidic side chains (eg, aspartic acid and glutamic acid), amino acids having uncharged polar side chains (eg, glycine). , Asparagine, glutamine, serine, threonine, tyrosine, cysteine and tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine and methionine), amino acids with beta-branched side chains (Eg, threonine, valine, and isoleucine) and amino acids having aromatic side chains (eg, tyrosine, phenylalanine, tryptophan and histidine). Therefore, one or more amino acid residues present in the CDR regions of the antibodies of the present invention may be substituted by other amino acid residues belonging to the same side chain group, and such modified antibodies function (i.e. , It can be tested as to whether or not the above functions (c) to (l) have been acquired.

Antibodies that bind to the same epitope as the anti-PD-1 antibody of the present invention

In another embodiment, the present invention provides an antibody that binds to an epitope on human PD-1 that is identical to the epitope of any of the PD-1 monoclonal antibodies of the invention (i.e., any of the monoclonal antibodies of the invention Antibodies and antibodies having the ability to compete with each other for PD-1 binding] are provided. In a preferred embodiment, the reference antibody used in the cross-competition study is monoclonal antibody 17D8 (an antibody having the V _H and V _L sequences shown in SEQ ID NO: 1 and SEQ ID NO: 8, respectively), or monoclonal antibody 2D3 (SEQ ID NO: _{An antibody having the V H} and V _L sequences shown in SEQ ID NO: 2 and SEQ ID NO: 9, respectively _{), or a monoclonal antibody 4H1 (an antibody having the V H} and V _L sequences shown in SEQ ID NO: 3 and SEQ ID NO: 10, respectively), or a monoclonal _{Rhonal antibody 5C4 (antibody having the V H} and V _L sequences shown in SEQ ID NO: 4 and SEQ ID NO: 11, respectively _{), or monoclonal antibody 4A11 (V H} and V _L sequences shown in SEQ ID NO: 5 and SEQ ID NO: 12, respectively) A branched antibody), or monoclonal antibody 7D3 (an antibody having the V _H and V _L sequences shown in SEQ ID NO: 6 and SEQ ID NO: 13, respectively), or monoclonal antibody 5F4 (V shown in SEQ ID NO: 7 and SEQ ID NO: 14, respectively) _H and V _L sequences). Such cross-competitive antibodies can be identified based on their ability to cross-compet with 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4 in a standard PD-1 assay. For example, Biacore assays, ELISA assays, or flow cytometry can be used to demonstrate the ability to compete with the antibodies of the invention. Through the ability of the test antibody to inhibit the binding of, for example, 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4 and human PD-1, this test antibody is 17D8, 2D3 for binding to human PD-1. , 4H1, 5C4, 4A11, 7D3 or 5F4, thus demonstrating that it can bind to an epitope on human PD-1, which is the same as that of 17D8, 2D3, 4H1, 5C4 or 4A11. In a preferred embodiment, the antibody that binds the epitope on human PD-1, which is the same as the epitope of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4, is a human monoclonal antibody. Such human monoclonal antibodies can be prepared and isolated as described in the Examples.

Engineered and Modified Antibodies

The antibody of the present invention _{may also be prepared using an antibody having one or more of the V H} and/or V _L sequences disclosed herein as a starting material for engineering the modified antibody, and the antibody thus modified is a starting antibody and Can have different (modified) properties. Antibodies can be engineered by modifying one or two variable regions (ie, V _H and/or V _L ), e.g., by modifying one or more residues present within one or more CDR regions and/or one or more framework regions. Additionally or alternatively, the antibody can be engineered to alter the effector function(s) of the antibody by modifying the residues in the constant region(s).

One type of variable region manipulation that can be performed is CDR grafting. Antibodies interact with target antigens primarily through amino acid residues present within the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences present in the CDRs are more diverse between individual antibodies compared to sequences outside the CDRs. Since CDR sequences are involved in most antibody-antigen interactions, by constructing an expression vector containing CDR sequences derived from a specific naturally occurring antibody grafted onto a framework sequence derived from different antibodies having different properties, specific natural Recombinant antibodies that simulate the properties of the resulting antibody can be expressed [eg, Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. et al. (1989) Proc. Natl. See Acad]. See also U.S.A. 86:10029-10033; US 5,225,539 (Winter), and US 5,530,101; 5,585,089; 5,693,762 and 6,180,370 (Queen et al.)].

Therefore, other embodiments of the present invention are SEQ ID NOs: 15, 16, 17, 18, 19, 20 and 21, SEQ ID NOs: 22, 23, 24, 25, 26, 27 and 28, respectively, and SEQ ID NOs: 29, 30, 31 , A heavy chain variable region comprising a CDR1, CDR2, and CDR3 sequence comprising an amino acid sequence selected from the group consisting of 32, 33, 34 and 35, and SEQ ID NOs: 36, 37, 38, 39, 40, 41 and 42, respectively, SEQ ID NOs: 43, 44, 45, 46, 47, 48 and 49, and SEQ ID NOs: 50, 51, 52, 53, 54, 55 and 56 CDR1, CDR2 and CDR3 sequences comprising an amino acid sequence selected from the group consisting of It relates to an isolated monoclonal antibody or antigen-binding portion thereof, comprising a light chain variable portion comprising. _{Therefore, these antibodies contain the V H} and V _L CDR sequences of monoclonal antibodies 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4, and may also contain different framework sequences derived from these antibodies.

Such framework sequences can be obtained from public DNA databases or published references containing germline antibody gene sequences. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the “VBase” human germline sequence database (www.mrc-cpe.cam.ac.uk/vbase) and Kabat, E. A et al. Multiple (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, IM et al. (1992) "The Repertoire of Human Germline V _H Sequences Reveals about Fifty Groups of V _H Segments with Different Hypervariable Loops" J. Mol. Biol. 227:776-798; And Cox, JPL et al. (1994) "A Directory of Human Germ-line V _H Segments Reveals a Strong Bias in their Usage" Eur. J. Immunol. 24:827-836; The contents of each document can be found in [which is clearly cited herein for reference. As another example, germline DNA sequences for human heavy and light chain variable region genes can be viewed in the GenBank database. For example, the following heavy chain germline sequence can be found in HCo7 HuMAb mice (GenBank accession numbers for this sequence are as follows: 1-69 (NG_0010109, NT_024637 and BC070333), 3-33 (NG_0010109 and NT_024637) And 3-7 (NG_0010109 and NT_024637)]. As another example, the following heavy chain germline sequence can be observed in HCo12 HuMAb mice (GenBank accession numbers for this sequence are as follows: 1-69 (NG_0010109, NT_024637 and BC070333), 5-51 (NG_0010109 and NT_024637) , 4-34 (NG_0010109 and NT_024637), 3- 30.3 (AJ556644) and 3-23 (AJ406678)].

Preferred framework sequences used in the antibodies of the present invention include sequences structurally similar to those used in the selected antibodies of the present invention, for example, the V _H 3-33 framework used by the preferred monoclonal antibodies of the present invention. Sequence similar to sequence (SEQ ID NO: 71) and/or V _H 4-39 frame sequence (SEQ ID NO: 73) and/or V _κ L6 frame sequence (SEQ ID NO: 72) and/or V _κ L15 frame sequence (SEQ ID NO: 74) There is this. The V _H CDR1, CDR2 and CDR3 sequences and the V _κ CDR1, CDR2 and CDR3 sequences may be grafted into a frame region having the same sequence as found in the germline immunoglobulin gene from which the frame sequence is derived, or the CDR sequence. Can be implanted on a frame site containing one or more mutations relative to the germline sequence. For example, in any case, it is advantageous to mutate residues present in the framework region to maintain or enhance the antigen-binding ability of the antibody [see, eg, US Pat. No. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 (Queen and many others)].

Another type of variable region modification is to improve one or more properties (e.g., affinity) of the antibody of interest by mutating amino acid residues present in the _{V H} and/or V _{κ CDR1, CDR2 and/or CDR3 regions.} There is something. Position-oriented mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and antigen binding efficacy, or other desired functional properties are as provided herein and in the Examples. It can be evaluated in the same in vitro or in vivo assay. It is desirable to introduce conservative modifications (as described above). The mutation may be amino acid substitution, addition or deletion, but substitution is preferred. Moreover, it is common for less than 1, 2, 3, 4 or 5 residues to be modified in the CDR region.

Therefore, in another embodiment, the present invention comprises (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20 and 21, or SEQ ID NOs: 15, 16, 17, 18, _{A V H} CDR1 region comprising an amino acid sequence comprising 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to the sequences of 19, 20 and 21; (b) an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 23, 24, 25, 26, 27 and 28, or one compared to the sequences of SEQ ID NOs: 22, 23, 24, 25, 26, 27 and 28 _{, A V H} CDR2 region comprising an amino acid sequence comprising 2, 3, 4 or 5 amino acid substitutions, deletions or additions; (c) an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34 and 35, or one compared to the sequence of SEQ ID NOs: 29, 30, 31, 32, 33, 34 and 35 _{, A V H} CDR3 region comprising an amino acid sequence comprising 2, 3, 4 or 5 amino acid substitutions, deletions or additions; (d) an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 37, 38, 39, 40, 41 and 42, or one compared to the sequence of SEQ ID NOs: 36, 37, 38, 39, 40, 41 and 42 _{, A V κ} CDR1 region comprising an amino acid sequence comprising 2, 3, 4 or 5 amino acid substitutions, deletions or additions; (e) an amino acid sequence selected from the group consisting of SEQ ID NOs: 43, 44, 45, 46, 47, 48 and 49, or one compared to the sequences of SEQ ID NOs: 43, 44, 45, 46, 47, 48 and 49 _{, A V κ} CDR2 region comprising an amino acid sequence comprising 2, 3, 4 or 5 amino acid substitutions, deletions or additions; And (f) an amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 51, 52, 53, 54, 55 and 56, or 1 compared to the sequences of SEQ ID NOs: 50, 51, 52, 53, 54, 55 and 56 An isolated anti-PD-1 monoclonal comprising a heavy chain variable region _{comprising a V κ} CDR3 region comprising an amino acid sequence comprising canine, 2, 3, 4 or 5 amino acid substitutions, deletions or additions Antibodies or antigen-binding portions thereof are provided.

Engineered antibodies of the present invention include antibodies in which the framework residues present in _{V H} and/or V _{κ are modified to improve the properties of the antibody.} Typically these framework modifications are applied to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, the antibody subjected to somatic mutation may contain framework residues different from the germline sequence from which the antibody is derived. These residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody was derived.

For example, Table 1 below shows a number of amino acid mutations occurring in the framework regions of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4, which are anti-PD-1 antibodies different from the heavy chain parental germline sequence. In order to restore one or more of the amino acid residues in the frame region sequence to its germline sequence, the somatic mutation is "reversed to the germline sequence by, for example, a position-oriented mutagenesis method or a PCR-mediated mutagenesis method. Can be "mutated".

Amino acid mutations can occur within the framework of the anti-PD-1 antibody that differs from the light chain parental germline sequence. For example, for 17D8, amino acid residue 47 (in FR2) of _{V κ} is isoleucine, whereas amino acid residue 47 present in the _{corresponding V κ L6 germline sequence is leucine.} In order to restore the frame region sequence to its germline sequence, the somatic mutation can be "back mutated" to the germline sequence, for example by position-oriented mutagenesis or PCR-mediated mutagenesis [ For example, _{residue 47 of V κ} of 17D8 (residue 13 of FR2) may be “reverse mutated” from isoleucine to leucine].

As another example, for 4A11, amino acid residue 20 (in FR1) of _{V κ} is serine, while amino acid residue 20 present in the _{corresponding V κ L15 germline sequence is threonine.} To restore the frame region sequence to its germline sequence, for example, _{residue 20 of V κ} of 4A11 can be “back mutated” from serine to threonine. Such “reverse mutated” antibodies are also included in the present invention.

_{The arrangement of the V H} regions of 17D8, 2D3, 4H1, 5C4 and 7D3 with respect to the parental germline V _H 3-33 sequence is shown in FIG. 8. _{The arrangement of the V H} regions of 4A11 and 5F4 with respect to the parental germline V _H 4-39 sequence is shown in FIG. 11.

Another type of framework modification involves mutating one or more residues present in the framework region or one or more CDR regions to eliminate T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This method is also referred to as "deimmunization" and is described in more detail in US Patent Publication 20030153043 (Carr et al.).

In addition to or as an alternative to the modifications made within the frame region or CDR region, the antibody of the present invention can be engineered to include modifications within the Fc region, as a result, typically one or more of the functional characteristics of the antibody. For example, it can denature serum half-life, complement fixation, Fc receptor binding and/or antigen-dependent intracellular cytotoxicity. In addition, the antibodies of the invention may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody), or the glycosylation of the antibody may be modified to denature one or more functional properties of the antibody. . Each of these specific examples is described in more detail below. The numbering method in the Fc region is according to the method of the EU index of Cabot.

In one embodiment, _{the hinge portion of C H1} is modified to alter (eg, increase or decrease) the number of cysteine residues present within the hinge portion. This method is described in more detail in US Pat. No. 5,677,425 (Bodmer et al.). The number of cysteine residues in the hinge portion of C _H1 is altered, for example, to promote assembly of light and heavy chains, or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge portion of the antibody is mutated to reduce the biological half-life of the antibody. More specifically, when one or more amino acid mutations are _{introduced into the C H2} -CH ₃ domain interface site of the Fc-hinge fragment, the antibody binds Staphylococcus protein A (SpA) compared to the original Fc-hinge domain SpA-binding ability. The ability is impaired. This method is described in more detail in US Pat. No. 6,165,745 (Ward et al.).

In another embodiment, an antibody of the invention is modified to increase its biological half-life. This can be done through various methods. For example, one or more of the following mutations may be introduced: T252L, T254S, T256F (described in US Pat. No. 6,277,375 (Ward)). Alternatively, to increase the biological half-life, the antibody is _{modified within the C H1} or C _L region, resulting in a salvage receptor binding epitope derived from the two loops _{of the C H2} domain of the Fc region of IgG. May contain [see US Patent Nos. 5,869,046 and 6,121,022 (Presta et al.)].

In another embodiment, the Fc region is modified by substituting one or more amino acid residues with different amino acid residues, resulting in altered effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 may be substituted with different amino acid residues, so that this antibody has an affinity for the effector ligand. It is denatured, but retains the antigen binding ability of the parent antibody. Effector ligands with altered affinity can be, for example, the C1 component of complement or an Fc receptor. This approach is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260 (Winter et al.).

In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 may be substituted with different amino acid residues, as a result of which the antibody has altered C1q binding capacity and/or complement dependent cytotoxicity (CDC). This can be reduced or eliminated. This approach is described in more detail in US Pat. No. 6,194,551 (Idusogie et al.).

In another example, one or more amino acid residues of amino acids at positions 231 and 239 are altered, thereby denaturing the antibody's ability to immobilize complement. This approach is described in more detail in PCT publication WO 94/29351 (Bodmer et al.).

In another example, the Fc region increases and/or increases the ability of the antibody to mediate antibody-dependent intracellular cytotoxicity (ADCC) by modifying one or more amino acids present at positions such as: Modified to increase the affinity of the antibody: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285 , 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333 , 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is described in more detail in PCT publication WO 00/42072 (Presta). Moreover, the binding sites for FcγR1, FcγRII, FcγRIII and FcRn, which are present on human IgG1, were mapped, and for variants with improved binding capacity, see Shields, R.L. et al. (2001) J. Biol. Chem. 276:6591-6604. Certain mutations occurring at positions 256, 290, 298, 333, 334 and 339 were found to improve the binding ability to FcγRIII. In addition, the following combination mutants were also found to improve the binding ability to FcγRIII: T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A.

In another embodiment, the glycosylation of the antibodies of the invention is modified. For example, antibodies that are not glycosylated (ie, antibodies that have not undergone glycosylation) can be produced. Glycosylation can be modified, for example, to increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be made, for example, by modifying one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions occur, so that one or more variable region glycosylation sites are removed, resulting in no glycosylation at these positions. Such glycosylation can increase the affinity of an antibody for an antigen. This approach is described in more detail in U.S. Patent Nos. 5,714,350 and 6,350,861 (Co et al.).

Additionally or alternatively, the antibody can be produced as an antibody in which a modified type of glycosylation occurs, e.g., a hypofucosylated antibody with a reduced amount of fucosyl residues or an antibody with an increased heterogeneous GlcNac structure. This modified glycosylation pattern serves as evidence demonstrating that the ADCC ability of the antibody was increased. Such carbohydrate modifications can be made, for example, by expressing the antibody in a host cell whose glycosylation machinery has been modified. Cells with modified glycosylation machinery are known in the art, and such cells can be used as host cells for expressing the recombinant antibodies of the present invention to produce antibodies with modified glycosylation. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (alpha(1,6) fucosyltransferase), so the antibodies expressed in the Ms704, Ms705 and Ms709 cell lines are its carbohydrates. Ingredients lack fucose. The Ms704, Ms705 and Ms709 FUT8 ^-/- cell lines were produced by targeting and destroying the FUT8 gene in CHO/DG44 cells using a 2 substitution vector [US Patent Publication 20040110704 (Yamane et al.) and Yamane-Ohnuki et al. (2004) ) Biotechnol Bioeng 87 :614-22]. As another example, EP 1,176,195 (Hanai et al.) describes a cell line in which the FUT8 gene encoding fucosyl transferase is functionally disrupted, wherein the antibody expressed in such cell line is alpha 1,6 binding-related Hypofucosylation occurs by reducing or eliminating enzymes. Hanai et al. also include a cell line with low enzymatic activity that binds to the Fc region of an antibody or adds fucose to N-acetylglucosamine that does not have an enzyme activity, for example, a rat myeloma cell line YB2/0 (ATCC CRL 1662) Has been described. PCT publication WO 03/035835 (Presta) describes a variant CHO cell line, Lec13 cells, with reduced ability to attach fucose to Asn(297)-binding carbohydrates, wherein the antibody expressed in the host cell Hypopocosylation occurs in [Shields, RL et al. (2002) J. Biol. Chem. 277:26733-26740]. In PCT Publication WO 99/54342 (Umana et al.), it is engineered to express a glycoprotein-modified glycosyl transferase (e.g., beta (1,4)-N-acetylglucosaminetransferase III (GnTIII)). Here, the antibody expressed in the engineered cell line increases the didifferentiated GlcNac structure, and as a result, the ADCC activity of the antibody is also increased [Umana et al. (1999) Nat. Biotech. 17: 176-180]. Alternatively, the fucose residues of the antibody can be cleaved off using a fucosidase enzyme. For example, fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies [Tarentino, AL. et al. (1975) Biochem. 14:5516-23].

Other modifications of the antibodies of the present application considered by the present invention include pegization. When an antibody is PEGylated, for example, the biological (eg, serum) half-life of the antibody can be increased. In order to peg an antibody, typically the antibody or fragment thereof is reacted with polyethylene glycol (PEG), such as an aldehyde derivative or reactive ester of PEG, under conditions such that one or more PEG groups are attached to the antibody or antibody fragment. Preferably, the PEGization is carried out through an acylation reaction or an alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to any type of PEG used to derivatize other proteins, such as mono(C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. It is meant to include. In certain embodiments, the antibody to be PEGylated is a non-glycosylated antibody. A method for PEGizing a protein is known in the art, and can be applied to the antibody of the present invention. See, for example, EP 0 154 316 (Nishimura et al.) and EP 0 401 384 (Ishikawa et al.).

How to manipulate antibodies

As described above, the anti-PD-1 antibody having the _{V H} and V _κ sequences disclosed herein is a novel anti-PD-1 antibody by modifying the _{V H} and/or V _{κ sequence or the constant region(s) attached thereto.} Can be used to produce Therefore, in another aspect of the invention, the structural features of the anti-PD-1 antibodies of the invention, e.g., 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4, are examples of one or more functional properties of the antibodies of the invention. For example, it is used to produce structurally related anti-PD-1 antibodies that retain the properties of binding to human PD-1. For example, one or more CDR regions of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4, or mutants thereof, are recombinantly linked with known framework regions and/or other CDRs, and the present invention as described above. Additional, recombinantly engineered, anti-PD-1 antibodies can be produced. Other types of variations include those described in the previous paragraph. In the engineering method, the starting material is _{one or more of the V H} and/or V _κ sequences provided herein, or one or more CDR regions thereof. In order to produce an engineered antibody, it is not necessary to substantially prepare (i.e., express as a protein) an antibody having one or more _{of the V H} and/or V _{κ sequences provided herein, or one or more CDR regions thereof.} Rather, the information contained in the sequence(s) is used as starting material to produce the "second generation" sequence(s) derived from the sequence of origin(s), and then this "second generation" sequence(s) is produced. Therefore, it will have to be expressed as a protein.

Therefore, in another embodiment, the present invention provides a method for producing an anti-PD-1 antibody comprising the following steps:

(a) (i) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20 and 21; A CDR2 sequence selected from the group consisting of SEQ ID NOs: 22, 23, 24, 25, 26, 27 and 28; And/or a heavy chain variable region antibody sequence comprising a CDR3 sequence selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34 and 35; And/or (ii) a CDR1 sequence selected from the group consisting of SEQ ID NOs: 36, 37, 38, 39, 40, 41 and 42; A CDR2 sequence selected from the group consisting of SEQ ID NOs: 43, 44, 45, 46, 47, 48 and 49; And/or providing a light chain variable region antibody sequence comprising a CDR3 sequence selected from the group consisting of SEQ ID NOs: 50, 51, 52, 53, 54, 55 and 56;

(b) modifying one or more amino acid residues present in the heavy chain variable region antibody sequence and/or light chain variable region antibody sequence to generate one or more modified antibody sequences; And

(c) expressing the modified antibody sequence as a protein.

Modified antibody sequences can be prepared and expressed using standard molecular biological techniques.

Preferably, the antibody encoded by the modified antibody sequence(s) is an antibody that retains one, some or all of the functional properties of the anti-PD-1 antibody disclosed herein, and its functional properties include: However, it is not limited thereto:

(a) the characteristic of binding to human PD-1 and K _D 1×10 ^{-7 M or less;}

(b) a property that does not substantially bind human CD28, CTLA-4 or ICOS;

(c) the property of increasing T cell proliferation in the MLR assay;

(d) the property of increasing the production of interferon-gamma in the MLR assay;

(e) the property of increasing the secretion of IL-2 in the MLR assay;

(f) properties of binding to human PD-1 and cynomolgus monkey PD-1;

(g) a property of inhibiting the binding of PD-L1 and/or PD-L2 to PD-1;

(h) properties that promote antigen-specific memory responses;

(i) properties that promote antibody response; And

(j) Properties that inhibit the growth of tumor cells in vivo.

The functional properties of the modified antibodies are known in the art and/or can be assessed using standard assays disclosed herein, such as the assays presented in the Examples (e.g., flow cytometry and binding assays).

In any embodiment of the antibody engineering method of the present invention, mutations can be introduced randomly or selectively over all or part of the anti-PD-1 antibody coding sequence, resulting in modified anti-PD- 1 Antibodies can be screened for binding activity and/or other functional properties as disclosed herein. Methods of mutation are known in the art. For example, PCT publication WO 02/092780 (Short) describes a method for screening antibody mutations by inducing antibody mutations using a saturation mutation induction method, a synthetic ligation assembly method, or a combination of these methods. Alternatively, PCT publication WO 03/074679 (Lazar et al.) discloses a method using a computer screening method to optimize the physicochemical properties of an antibody.

Nucleic Acid Molecules Encoding Antibodies of the Invention

Another aspect of the invention relates to a nucleic acid molecule encoding an antibody of the invention. The nucleic acid may be present in whole cells or cell lysates, or may be present in a specifically purified or substantially pure form. Nucleic acid can be used for other cellular components or other contaminants such as other cellular nucleic acids or proteins as standard techniques such as alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other sugars. A nucleic acid "isolated" or "substantially pure" when purified and removed by methods well known in the art. [F. Ausubel et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acid of the present invention may be, for example, DNA or RNA, and may or may not contain an intron sequence. In a preferred embodiment, the nucleic acid is a cDNA molecule.

The nucleic acids of the present invention can be obtained using standard molecular biological techniques. In the antibody expressed by a hybridoma [a hybridoma produced from a transgenic mouse carrying a human immunoglobulin gene, as described in more detail below], the light and heavy chains of the antibody produced by the hybridoma CDNA encoding can be obtained by standard PCR amplification method or cDNA cloning technique. In the antibody obtained by the immunoglobulin gene library (eg, using phage display technology), the nucleic acid encoding the antibody can be recovered from the library.

_{Preferred nucleic acid molecules of the invention are those encoding the V H} and V _L sequences of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 monoclonal antibodies. The 17D8, 2D3, 4H1, 5C4, 4A11, shows the DNA sequence encoding the V _H sequences of the 5F4 and 7D3 in respective SEQ ID NO: 57, 58, 59, 60, 61, 62 and 63. _{DNA sequences encoding the V L} sequences of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are shown in SEQ ID NOs: 64, 65, 66, 67, 68, 69 and 70, respectively.

Once _{the DNA fragments encoding the V H} and V _L segments are obtained, such DNA fragments are further manipulated, for example by standard recombinant DNA techniques, resulting in variable region genes being converted to full-length antibody chain genes, Fab fragments. Gene or scFv gene. In such an engineering process, the V _L -or V _H -encoding DNA fragment is operably linked to another protein such as an antibody constant region or other DNA fragment encoding a flexible linker. In the present specification, the term "operably linked" refers to a case in which two DNA fragments are joined so that the amino acid sequence encoded by the two DNA fragments remains in an in-frame state. .

The isolated DNA encoding the _{V H} site can be converted into a full-length heavy chain gene by operably linking the _{V H} -encoding DNA to _{other DNA molecules encoding the heavy chain constant regions (C H1} , C _H2 and CH _{3 ).} The sequence of human heavy chain constant region genes is known in the art (e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. . 91-3242], DNA fragments containing these sites can be obtained by standard PCR amplification methods. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably an IgG1 or IgG4 constant region. For Fab fragment heavy chain genes, the V _H -encoding DNA can be operably linked to other DNA molecules encoding only the heavy chain C _{H1 constant region.}

The isolated DNA encoding the _{V L} region can be converted into a full-length light chain gene (and Fab light chain gene) by operably linking the _{V L} -encoding DNA to another DNA molecule encoding the light chain constant region C _L. The sequence of the human light chain constant region gene is known in the art (for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. . 91-3242], DNA fragments containing such sites can be obtained by standard PCR amplification methods. The light chain constant region may be a kappa or lambda constant region, with the kappa constant region most preferred.

To produce the scFv gene, the V _H -and V _L -encoding DNA fragments can be operably linked to a flexible linker, e.g., _{other fragments encoding the amino acid sequence (Gly 4} -Ser) _{3, resulting in} , V _H and V _{L sequences can be expressed as a contiguous single-chain protein having V L} and V _H sites linked by a flexible linker (eg, Bird et al. (1988) Science 242:423- 426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554].

Production of monoclonal antibodies of the invention

The monoclonal antibody (mAb) of the present invention is prepared by various techniques, such as conventional monoclonal antibody methods, for example, by standard somatic cell hybridization techniques according to Kohler and Milstein (1975) Nature 256: 495. Can be produced. Although somatic cell hybridization methods are preferred, in principle, other techniques for producing monoclonal antibodies, such as viral transformation of B lymphocytes or oncogene transformation, can also be used.

As a preferred animal system for producing hybridomas, there is a murine system. Methods for producing hybridomas in mice are well established. Immunization procedures and techniques for isolating immunized splenocytes for fusion are also known in the art. Fusion partners (eg, murine myeloma cells) and methods of fusion are also known.

The chimeric or humanized antibody of the present invention can be prepared based on the sequence of a murine monoclonal antibody prepared as described above. DNA encoding heavy and light chain immunoglobulins is obtained from the murine hybridoma of interest using standard molecular biological techniques to contain the immunoglobulin sequence of a non-murine animal (e.g., human). Can be manipulated. For example, in order to prepare a chimeric antibody, the murine variable region can be bound to the human constant region using a method known in the art (see, for example, US Pat. No. 4,816,567 (Cabilly et al.). ]. To produce humanized antibodies, murine CDR regions can be inserted into human frameworks using methods known in the art (e.g., US Pat. No. 5,225,539 (Winter) and US Pat. No. 5,530,101. arc; 5,585,089; 5,693,762 and 6,180,370 (Queen and many others).

In a preferred embodiment, the antibody of the invention is a human monoclonal antibody. The human monoclonal antibody produced against PD-1 as described above can be produced using transgenic or transchromosomal mice bearing a part of the human immune system rather than the mouse system. Such transgenic and trans chromosomal mice include mice referred to herein as HuMAb mice and KM mice™, respectively, and these are collectively referred to herein as "human Ig mice".

The HuMAb mice® (Medarex, Inc.) are human immunity encoding unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, with targeted mutations that inactivate the position of endogenous μ and κ chains. It contains miniloci of the globulin gene (see, eg, Lonberg et al. (1994) Nature 368(6474): 856-859). Therefore, in mice, the expression of mouse IgM or κ is reduced, and as immunized, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation, resulting in high affinity human IgGκ monoclonal. Antibodies are produced [Lonberg, N. et al. (1994) homologous; Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. NY. Acad. Sci. 764:536-546)]. Methods and uses of HuMAb mice, as well as genetic modifications performed by such mice, are described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Taylor, L. et al. (1994) International Immunology 6: 579-591; And Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851; The contents of these documents are as such are described in more detail in [incorporated herein by reference]. See also US Patent No. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; And 5,770,429 (all above, Lonberg et al.); US Patent No. 5,545,807 (Surani et al.); PCT publications WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962 (Lonberg and Kay); See also PCT Publication WO 01/14424 (all Korman et al.).

In another embodiment, the human antibody of the invention can be generated using a mouse carrying a human immunoglobulin sequence on a transgene and a trans chromosome, e.g., a mouse carrying a human heavy chain transgene and a human light chain trans chromosome. have. Such mice (referred to herein as "KM Mouse™") are described in detail in PCT Publication WO 02/43478 (Ishida et al.).

In addition, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to generate anti-PD-1 antibodies of the invention. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can be used; Regarding such mice, see, eg, US Pat. No. 5,939,598; 6,075,181; 6,114,598; It is described in 6,150,584 and 6,162,963 (Kucherlapati et al.).

In addition, alternative transchromosomal animal systems expressing human immunoglobulin genes are available in the art, which can be used to generate anti-PD-1 antibodies of the present invention. For example, mice carrying both human heavy chain transchromosomes and human light chain transchromosomes (referred to as “TC mice”) can be used; Regarding these mice, Tomizuka et al. (2000) Proc. Natl. Acad. Sd. USA 97:722-727. In addition, cows carrying human heavy and light chain trans chromosomes are known in the art [Kuroiwa et al. (2002) Nature Biotechnology 20:889-894], which is an anti-PD-1 antibody of the present invention. Can be used to create.

Human monoclonal antibodies of the invention can also be prepared using phage display methods for screening human immunoglobulin gene libraries. Such phage display methods for isolating human antibodies are established in the art. See, eg, US Pat. No. 5,223,409; 5,403,484; And 5,571,698 (Ladner et al.); U.S. Patent Nos. 5,427,908 and 5,580,717 (Dower et al.); U.S. Patent Nos. 5,969,108 and 6,172,197 (McCafferty et al.); And U.S. Patent No. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 (Griffiths et al.)].

The human monoclonal antibody of the present invention may also be prepared using SCID mice, that is, mice in which human immune cells are reconstituted and introduced, thereby allowing a human antibody response to occur when immunized. Such mice are described, for example, in U.S. Patent Nos. 5,476,996 and 5,698,767 (Wilson et al.).

Immunization of human Ig mice

When human Ig mice are used to generate the human antibodies of the present invention, these mice can be immunized using a purification or amplification agent of the PD-1 antigen and/or recombinant PD-1 or PD-1 fusion protein [Lonberg, N. et al. (1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851; And PCT publications WO 98/24884 and WO 01/14424]. The age of the mice at the time of the first injection will preferably be 6 to 16 weeks of age. For example, purified or recombinant preparations (5-50 μg) of the PD-1 antigen can be used to immunize human Ig mice intraperitoneally.

A method for producing a monoclonal antibody against PD-1, which is entirely human, is described in detail in Example 1 below. As a result of multiple immunizations with various antigens, transgenic mice were first immunized in peritoneal (IP) with antigens in complete Freund's adjuvants, then once every two weeks with antigens in incomplete Freund's adjuvants. It was found that the reaction was shown when IP immunization was performed (up to 6 times in total). However, adjuvants other than Freund's adjuvants have also been found to be effective. In addition, it was found that all cells are highly immunogenic in the absence of adjuvants. Immune responses can be observed throughout the course of immunization using plasma samples obtained through posterior orbital blood sampling. Plasma can be screened by ELISA (described below), and mice to which anti-PD-1 human immunoglobulin has been sufficiently added dropwise can be used for fusion. Three days before the mouse is euthanized and the spleen is isolated, intravenous injection of the antigen can strengthen the mouse's immune system. It is expected that 2-3 fusions will be required per immunization. Typically 6 to 24 mice are required per each immunization with the antigen. In general, both HCo7 and HCo12 variants are used. In addition, both HCo7 and HCo12 transgenes can be introduced together in one mouse (HCo7/HCo12) with two different human heavy chain transgenes. Alternatively or additionally, as described in Example 1, the KM Mouse™ variant can be used.

Preparation of hybridoma producing human monoclonal antibody of the present invention

In order to prepare a hybridoma producing the human monoclonal antibody of the present invention, splenocytes and/or lymph node cells derived from immunized mice are isolated, and this is an appropriate infinitely proliferating cell line, such as a mouse myeloma cell line. Can be fused to. The resulting hybridoma can be screened for producing antigen-specific antibodies. For example, a single cell suspension of splenic lymphocytes from immunized mice can be fused with 50% PEG to 1/6 of P3X63-Ag8.653 non-secreting mouse myeloma cells (ATCC, CRL 1580). Alternatively, single cell suspensions of splenic lymphocytes from immunized mice are fused via an electric field-based electrofusion method, using a Cyto Pulse large-chamber cell fusion electroporation system (Cyto Pulse Sciences, Inc., Glen Burnie, MD). Can be. Cells were plated on a flat bottom microtiter plate at a density of about 2×10 ⁵ and then 20% bovine clone serum, 18% “653” conditioning medium, 5% Origen (IGEN), 4 mM L-glutamine, 1 mM Sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamicin and 1X HAT (Sigma; HAT is added 24 hours after fusion) Incubated for 2 weeks in a selective medium containing After about 2 weeks, the cells can be cultured in a medium in which HAT is changed to HT. Each well can then be screened for the presence of human monoclonal IgM and IgG antibodies by ELISA. Once excess hybridoma growth occurs, the medium can generally be observed after 10-14 days. Antibody secreting hybridomas can be replated for screening, and if still positive for human IgG, this monoclonal antibody can be subcloned two or more times by limiting dilution. Then, by culturing the stable subclones in vitro, a small amount of antibodies can be produced in the tissue culture medium and characterized.

In order to purify the human monoclonal antibody, the selected hybridoma can be grown in a 2 L spinner flask to purify the monoclonal antibody. The supernatant can be filtered and concentrated prior to affinity chromatography using protein A-Sepharose (Pharmacia, Piscataway, NJ). Purity can be confirmed by checking the eluted IgG through gel electrophoresis and high performance liquid chromatography. The buffer solution is changed to PBS, and the concentration can be measured through _{OD 280 when using a 1.43 extinction coefficient.} Monoclonal antibodies can be aliquoted and stored at -80°C.

Preparation of transfectoma producing monoclonal antibody of the present invention

Antibodies of the present invention can also be produced in host cell transfectomas, for example, by combining recombinant DNA technology and gene transfection methods as well known in the art [eg, Morrison, S. (1985) Science 229:1202].

For example, in order to express an antibody or an antibody fragment thereof, DNA encoding the partial or full length light and heavy chains is prepared by standard molecular biological techniques (e.g., PCR amplification method using hybridomas expressing the antibody of interest. Or cDNA cloning method), and the DNA can be inserted into an expression vector so that this gene can be operably linked with transcription and translation control sequences. Here, the term "operably linked" refers to a case in which an antibody gene is ligated to a vector, so that the transcription and translation control sequences in the vector can control the transcription and translation of the antibody gene. The expression vector and expression control sequence are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene may be inserted into separate vectors, and more typically, the two genes are inserted into the same vector. The antibody gene is inserted into the expression vector by standard methods (eg, a method of ligation of an antibody gene fragment with a complementary restriction site present on the vector, or a blunt end ligation method if no restriction site is present). The light chain and heavy chain variable regions of the antibodies described herein are inserted into an expression vector capable of encoding the full-length antibody gene, the heavy chain constant region and the light chain constant region of the desired same reference sample antibody, and this V _H segment is a CH segment in the vector. By making operably associated with the (s) and making the V _{κ segment operably associated with the C L} segment in the vector, it can be used to produce a full-length antibody gene of any antibody homologous reference sample type. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that promotes secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into a vector, and as a result, the signal peptide is associated in-frame with the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide derived from a non-immunoglobulin protein).

In addition to the antibody chain gene, the recombinant expression vector of the present invention carries regulatory sequences that control the expression of the antibody chain gene in the host cell. The term "regulatory sequence" is meant to include promoters, enhancers and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of antibody chain genes. Regarding these regulatory sequences, see, eg, Goeddel, Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990). One skilled in the art said that the design of the expression vector, including the selection of regulatory sequences, may vary depending on factors such as the selection of the transformed host cell and the level of expression of the desired protein. Preferred regulatory sequences for expression of mammalian host cells include viral elements that increase the level of protein expression in mammalian cells, such as cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g. , Adenovirus major late promoter (AdMLP) and promoters and/or enhancers derived from polyoma virus Alternatively, non-viral regulatory sequences such as ubiquitin promoter or β-globin promoter can be used. In addition, regulatory elements composed of sequences derived from different sources, such as the SRα promoter system, i.e., a promoter system containing sequences derived from the long terminal repeats of the SV40 early promoter and type 1 human T cell leukemia virus may also be used. Can [Takebe, Y. et al. (1988) Mol. Cell. Biol. 8 :466-472].

In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may contain additional sequences, such as sequences that regulate the replication of the vector in the host cell (e.g., origin of replication) and selectable marker genes . The selection marker gene facilitates selection of host cells into which the vector has been introduced (see, eg, US Pat. Nos. 4,399,216, 4,634,665 and 5,179,017 (all patents by Axel et al.)). For example, a selectable marker gene typically confers resistance to drugs such as G418, hygromycin or methotrexate to the host cell into which the vector has been introduced. Includes ^- (used in dhfr host cells used for methotrexate selection / amplification) and the neo gene (used for G418 selection), a preferred selection marker gene as dihydro-reductase (DHFR) gene.

For the expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection method" include various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium-phosphate precipitation, DEAE-dextran traits. It means including infection method. Although theoretically the antibody of the present invention can be expressed in prokaryotic or eukaryotic host cells, such eukaryotic cells, specifically mammalian cells, are more appropriately folded than prokaryotic cells and assemble and secrete immunologically active antibodies Since it is easy to do so, expression of the antibody in such eukaryotic cells, particularly in mammalian host cells, is most preferred. Prokaryotic expression of antibody genes has been reported to be inefficient in producing active antibodies in high yield [Boss, MA and Wood, CR (1985) Immunology Today 6 :12-13].

Preferred mammalian host cells for expressing the recombinant antibody of the present invention include Chinese hamster ovary cells (CHO cells) [dhfr-CHO cells (Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220) ); DHFR selection markers (for example, use with R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621)], NSO myeloma cells, COS cells and SP2 cells. Specifically, the GS gene expression system (WO 87/04462, WO 89/01036 and EP 338,841) is another preferred expression system for use with NSO myeloma cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, sufficient time for the antibody to be expressed in the host cell, more preferably sufficient for secretion of the antibody in the culture medium in which the host cell is growing. For a period of time, the antibody can be produced by culturing the host cell. Antibodies can be recovered from the culture medium using standard protein purification methods.

Characterization of antibody and antigen binding

Antibodies of the present invention can be tested for binding to PD-1, for example by a conventional ELISA. Briefly, microtiter plates are coated with purified PD-1 (0.25 μg/ml in PBS) and then blocked with 5% bovine serum albumin in PBS. An antibody dilution (eg, a plasma dilution derived from a PD-1 immunized mouse) is added to each well and incubated at 37° C. for 1-2 hours. After washing the plate with PBS/Tween, 37° C. with a secondary reagent conjugated to alkaline phosphatase (eg, goat-anti-human IgG Fc-specific polyclonal reagent in the case of human antibodies). Incubate for 1 hour at. After washing, the plate was developed with pNPP substrate (1 mg/ml), which was then analyzed at OD 405-650. Preferably, mice showing the highest titer will be used for fusion.

The ELISA assay as described above can also be used to screen for hybridomas that test positive with the PD-1 immunogen. Hybridomas that bind PD-1 with high avidity are subcloned and then re-characterized. One clone derived from each hybridoma that retains parental reactivity (determined by ELISA) is selected, and a bank of 5 to 10 viable cells stored at -140°C can be made and used for antibody purification.

To purify anti-PD-1 antibodies, selected hybridomas can be grown in 2 L spinner-flasks for monoclonal antibody purification. Prior to affinity chromatography using Protein A-Sepharose (Pharmacia, Piscataway, NJ), the supernatant may be filtered and concentrated. The eluted IgG can be checked through gel electrophoresis and high performance liquid chromatography to confirm the purity. The buffer solution is changed to PBS and the concentration can be measured via _{OD 280 when using the 1.43 extinction coefficient.} Monoclonal antibodies can be aliquoted and stored at -80°C.

To determine whether the selected anti-PD-1 monoclonal antibody binds to a unique epitope, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). As described above, by using PD-1 coated ELISA plates, competition studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be conducted. Binding of biotinylated mAbs can be confirmed using a strept-avidin-alkaline phosphatase probe.

In order to measure the same reference sample type of the purified antibody, the same reference sample ELISA may be performed using a reagent specific to a specific same reference sample antibody. For example, to determine the same reference specimen type of human monoclonal antibody, wells of a microtiter plate can be coated with 1 μg/ml of anti-human immunoglobulin overnight at 4°C. After blocking with 1% BSA, the plate is reacted with a test monoclonal antibody of less than 1 μg/ml or a purified same reference sample control at room temperature for 1-2 hours. The wells can then be reacted with human IgG1 or human IgM-specific alkaline phosphatase-conjugated probes. As described above, the plate is developed and analyzed.

Anti-PD-1 human IgG can be tested for reactivity with PD-1 antigen by Western blotting. Briefly, PD-1 can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the isolated antigen is transferred to a nitrocellulose membrane, blocked with 10% fetal bovine serum, and then probed with the monoclonal antibody to be tested. Human IgG binding can be confirmed using anti-human IgG alkaline phosphatase and developed with BCIP/NBT substrate purification (Sigma Chem. Co., St. Louis, Mo.).

Immunoconjugate

In another aspect, the invention features an anti-PD-1 antibody or fragment thereof conjugated to a therapeutic moiety, such as a cytotoxin, drug (e.g., immunosuppressant) or radiotoxin. . Such conjugates are referred to herein as "immunoconjugates". Immunoconjugates containing one or more cytotoxins are referred to as “immunotoxins”. Cytotoxic or cytotoxic agents include any agents that are harmful to the cells (eg, kill cells). Examples thereof include Taxol, Cytocalacin B, Gramicidine D, Ethidium bromide, Emethine, Mitomycin, Etoposide, Tenofoside, Vincristine, Vinblastine, Colchicine, Doxorubicin, Donorubicin, Dihydroxy Anthracene dione, mitoxantrone, mithramycin, actinomycin D, 1-dihydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol and puromycin and analogs or homologs thereof. In addition, therapeutic agents include, for example, metabolic antagonists (e.g. methotrexate, 6-mercaptopurine, 6-thioguanine, citrabine, 5-fluorouracil dicarbazine), alkylating agents (e.g. meclo Retamine, Thioepa Chlorambucil, Melfaran, Carmustine (BSNU) and Romustine (CCNU), Cyclotosphamide, Busulfan, Dibromomannitol, Streptozotocin, Mitomycin C and cis-dichlorodiamine Platinum(II) (DDP) cisplatin), anthracyclines (e.g., donorubicin (formerly donomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin) And anthramycin (AMC)), and anti-mitotic agents (eg, vincristine and vinblastine).

Other preferred examples of therapeutic cytotoxins that can be conjugated to the antibody of the present invention include duocarmycin, calicheamicin, maytansine and auristatin and derivatives thereof. Examples of calicheamicin antibody conjugates are those that are commercially available (Mylotarg™; Wyeth-Ayerst).

Cytotoxins can be conjugated to the antibodies of the invention using linker techniques known in the art. Examples of linkers used to conjugate cytotoxins to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. The linker is sensitive to cleavage by low pH in the lysosomal compartment, or proteolytic enzymes, such as proteolytic enzymes that are preferentially expressed in tumor tissue, such as cathepsins (e.g., cathepsins B, C, It can be chosen to be susceptible to degradation by D).

The types and linkers of cytotoxins used to conjugate therapeutic agents to antibodies, and such methods are described in Saito, G. et al. (2003) Adv. Drug Deliv. Rev. 55:199-215; Trail, P.A. et al. (2003) Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell 3:207-212; Allen, T.M. (2002) Nat. Rev. Cancer 2:750-763; Pastan, I. and Kreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3:1089-1091; Senter, P.D. and Springer, CJ. (2001) Adv. Drug Deliv. Rev. 53:247-264.

Antibodies of the present invention can also be conjugated to radioactive isotopes to produce cytotoxic radiopharmaceuticals (also referred to as radioactive immunoconjugates). Examples of radioactive isotopes that can be conjugated to an antibody for diagnostic or therapeutic use include, but are not limited to, ^{iodine 131} , indium ¹¹¹ , yttrium ⁹⁰ and lutetium ^177. Methods of making radioactive immunoconjugates are established in the art. Examples of radioactive immunoconjugates include commercially available ones, such as Zevalin™ (IDEC Pharmaceuticals) and Bexxar™ (Corixa Pharmaceuticals), and by using the antibody of the present invention through a similar method, radioactive immunoconjugates You can also manufacture.

The antibody conjugates of the present invention can be used to modify certain biological reactions, and the drug portion should not be construed as limited to conventional chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide having a desired biological activity. Such proteins include, for example, enzymatic toxins or active fragments thereof such as abrin, lysine A, Pseudomonas exotoxin or diphtheria toxin; Proteins such as tumor necrosis factor or interferon-γ; Or bioreaction modifiers such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulation Factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moieties to antibodies are well known [eg, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds. ), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982)].

Bispecific molecule

In another aspect, the invention features a bispecific molecule comprising an anti-PD-1 antibody or fragment thereof of the invention. An antibody or antigen-binding portion thereof of the invention may be derivatized or bound to other functional molecules, such as other peptides or proteins (e.g., other antibodies or ligands for receptors), as a result of which, Bispecific molecules can be generated that bind to two or more different binding sites or target molecules. In fact, the antibodies of the invention can be derivatized or bound to one or more other functional molecules, resulting in multispecific molecules that bind to two or more different binding sites and/or target molecules; Such multispecific molecules are also included in the term "bispecific molecule" as used herein. In order to generate the bispecific molecules of the invention, the antibodies of the invention (e.g., by chemical coupling, genetic fusion or non-covalent binding, etc.) one or more other binding molecules, such as other antibodies, antibody fragments, It can be functionally bound to the peptide or binding mimetic, resulting in a bispecific molecule.

Therefore, the present invention includes a bispecific molecule comprising at least one first binding specificity factor for PD-1 and a second binding specificity factor for a second target epitope. In certain embodiments of the invention, the second target epitope is an Fc receptor such as human FcγRI (CD64) or human Fcα receptor (CD89). Therefore, the present invention is a dual specific molecule capable of binding both FcγR or human Fcα expression effector cells (e.g., monocytes, macrophages or polymorphonuclear cells (PMN)) and target cells expressing PD-1. Includes. These bispecific molecules target PD-1 expressing cells to effector cells, e.g., phagocytosis of PD-1 expressing cells, antibody dependent cell-mediated cytotoxicity (ADCC), cytokine release or superoxide anion. Triggers Fc receptor-mediated effector cell activity such as the production of.

In an embodiment of the present invention in which the bispecific molecule is also multispecific, the molecule may contain a third binding specificity factor in addition to the anti-Fc binding specificity factor and the anti-PD-1 binding specificity factor. In one embodiment, the third binding specificity factor is a molecule that binds to an anti-enhancing factor (EF) moiety, such as a surface protein involved in cytotoxic activity, thereby increasing the immune response against the target cell. "Anti-enhancement factor portion" is an antibody, functional antibody fragment, or antibody that binds to a predetermined molecule, such as an antigen or a receptor, and enhances the effect of the binding determinant on an Fc receptor or a target cell antigen. It can be a ligand. The “anti-enhancing factor moiety” is capable of binding to an Fc receptor or a target cell antigen. Alternatively, the anti-enhancing factor moiety may bind to an entity different from the entity to which the first and second binding specificity factors bind. For example, the anti-enhancing factor moiety is directed to cytotoxic T cells (e.g., via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cells that increase the immune response to target cells. ) Can be combined.

In one embodiment, the bispecific molecule of the invention comprises one or more antibodies, or antibody fragments thereof, such as Fab, Fab', F(ab') ₂ , Fv or single chain Fv as binding specificity factors. . Antibodies may also be light chain or heavy chain dimers, or may be any minimal fragment thereof, such as Fv or a single chain structure [Ladner et al., US Pat. No. 4,946,778; The content of this is cited for reference].

In one embodiment, the binding specificity for the Fcγ receptor is provided by a monoclonal antibody, the ability of which is not blocked by human immunoglobulin G (IgG). As used herein, the term “IgG receptor” refers to any of the eight γ-chain genes located on chromosome 1. These genes consist of a total of 12 transmembrane or soluble receptor subtypes [divided into 3 Fcγ receptor groups; FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD 16)]. In one preferred embodiment, the Fcγ receptor is a human high affinity FcγRI. Human FcγRI is a molecule of 72 kDa and has a high affinity for monomeric IgG (10 ^{8 -10 9} M ^-1 ).

For the production and characterization of any preferred anti-Fcγ monoclonal antibody see Fanger et al., PCT Publication WO 88/00052 and US Pat. No. 4,954,617; Its teachings are set forth in [incorporated herein by reference]. Since these antibodies bind to the epitope of FcγRI, FcγRII or FcγRIII at a position distinct from the Fcγ binding site of the receptor, their binding is not substantially blocked by physiological levels of IgG. Specific anti-FcγRI antibodies useful in the present invention include mAb22, mAb32, mAb44, mAb62 and mAb197. Hybridomas producing mAb32 can be obtained from the U.S. Parenthesis Culture Collection (ATCC accession number = HB9469). In other embodiments, the anti-Fcγ receptor antibody is a humanized form of monoclonal antibody 22 (H22). For the production and characterization of H22 antibodies, see Graziano, R.F. et al. (1995); J. Immunol 155 (10): 4996-5002 and PCT publication WO 94/10332. The H22 antibody-producing cell line has been deposited with the U.S. parentheses culture collection, the deposit name is HA022CL1 and the deposit number is CRL 11177.

In other preferred embodiments, the binding specificity for the Fc receptor is provided by an antibody that binds to a human IgA receptor, such as an Fc-alpha receptor (FcαRI (CD89)), the binding of which is provided by human immunoglobulin A (IgA). It is preferably blocked by. The term "IgA receptor" is meant to include the gene product (FcαRI) of one α-gene located on chromosome 19. This gene is known to alternatively encode a spliced dural subtype (55-110 kDa). FcαRI (CD89) is constitutively expressed on monocytes/macrophages, eosinophils and neutrophil granulocytes, but not on non-effector cell populations. The median affinity of FcαRI for both IgA1 and IgA2 (about 5×10 ⁷ M ⁻¹ ) increases when exposed to cytokines such as G-CSF or GM-CSF [Morton, HC et al. 1996) Critical Reviews in Immunology 16:423-440]. Four FcαRI-specific monoclonal antibodies (A3, A59, A62 and A77) that bind to FcαRI outside the IgA ligand binding domain are described in Monteiro, RC et al. (1992) J. Immunol. 148:1764].

FcαRI and FcγRI are preferred trigger receptors for use in the dual specific molecules of the present invention, because these receptors are (1) mainly immune effector cells such as monocytes, PMNs, macrophages and dendritic cells. Is expressed in; (2) expressed at high levels (eg, 5,000-100,000 per cell); (3) is a mediator of cytotoxic activity (eg ADCC, phagocytosis); (4) This is because it mediates the enhancement of the antigen-presenting action of antigens, including self-antigens targeted to this receptor.

When human monoclonal antibodies are desired, other antibodies that can be used in the dual specific molecules of the invention include murine, chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present invention can be prepared by conjugating constitutive binding specificity factors such as anti-FcR and anti-PD-1 binding specificity factors using methods known in the art. For example, each binding specificity factor of a bispecific molecule can be produced separately and then conjugated to each other. When it is a binding specificity factor group protein or peptide, various coupling agents or crosslinking agents can be used for covalent conjugation. Examples of crosslinking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide ( oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC ) [Eg, Karpovsky et al. (1984) J Exp. Med. 160: 1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648]. For other methods, see Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie et al. (1987) J Immunol. 139: 2367-2375. Preferred conjugating agents include SATA and sulfo-SMCC, all of which are commercially available from Pierce Chemical Co. (Rockford, IL).

When the binding specificity factor is an antibody, it can be conjugated through a sulfhydryl bond of the C-terminal hinge portion of the two heavy chains. In a particularly preferred embodiment, the hinge portion can be modified to contain an odd number, preferably one sulfhydryl moiety, prior to conjugation.

Alternatively, the two binding specificity factors can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the dual specific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab') ₂ or ligand x Fab fusion protein. The bispecific molecule of the present invention may be a single chain molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific molecules may contain two or more single chain molecules. Methods of making bispecific molecules are described in US Pat. Nos. 5,260,203; US Patent No. 5,455,030; US Patent No. 4,881,175; US Patent No. 5,132,405; US Patent No. 5,091,513; US Patent No. 5,476,786; US Patent No. 5,013,653; US Patent No. 5,258,498; And US Patent No. 5,482,858.

The ability of a bispecific molecule to bind its specific target is determined by, for example, enzyme-linked immunosorbent assay (ELISA), radioactive immunoassay (RIA), FACS assay, bioassay (e.g., growth inhibition) or Western It can be confirmed by a blot assay. Each of these assays generally detects the presence of a protein-antibody complex having a specific purpose using a labeling reagent (eg, an antibody) specific for the complex of interest. For example, the FcR-antibody complex can be detected using, for example, an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to the antibody-FcR complex. Alternatively, the complex can be detected using any of a variety of other immunoassays. For example, antibodies can be radiolabeled and used in radioactive immunoassays (RIA) (e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, incorporated herein by reference. The Endocrine Society, March, 1986]. Radioactive isotopes can be detected using a γ counter or scintillation counter or radiographic method.

Pharmaceutical composition

In another aspect, the present invention comprises a monoclonal antibody of the present invention or an antigen-binding portion thereof, or comprises a combination thereof, for example, a composition prepared with a pharmaceutically acceptable carrier, e.g., Provides a pharmaceutical composition. Such compositions may comprise one or a combination of the antibodies or immunoconjugates or bispecific molecules of the invention or these (eg, two or more different ones). For example, the pharmaceutical composition of the present invention may contain antibodies (or immunoconjugates or bispecific antibodies) that bind to different epitopes on a target antigen or have complementary activity in combination.

The pharmaceutical composition of the present invention may also be administered as a combination therapy, ie in combination with other agents. For example, the combination therapy may include both one or more other anti-inflammatory or immunosuppressive agents and an anti-PD-1 antibody of the invention. Examples of therapeutic agents that can be used in combination therapy are described in more detail in the section below regarding the use of the antibodies of the invention.

As used herein, “pharmaceutically acceptable carrier” includes any and all of physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epithelial administration (eg, administration by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, immunoconjugate or bispecific molecule can be coated with any material, which will protect the compound from the action of acids and other natural conditions that can inactivate the compound. I can.

The pharmaceutical composition of the present invention may contain one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound, but does not confer undesirable toxicological effects (eg, Berge, SM et al. (1977) J. Pharm). . Sci. 66:1-19]. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include salts derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, iodic hydride and phosphorus, and non-toxic organic acids such as aliphatic mono-carboxylic acids and dicarboxylic acids. And salts derived from carboxylic acids, phenyl-substituted alkanonic acids, hydroxy alkanonic acids, aromatic acids, aliphatic acids and aromatic sulfuric acids, and the like. As the base addition salt, salts derived from alkaline earth metals such as sodium, potassium, magnesium and calcium, and non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, and chloro And salts derived from procaine, choline, diethanolamine, ethylenediamine, and procaine.

The pharmaceutical composition of the present invention may also contain a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfate, sodium metabisulfite and sodium sulfite; (2) Fat-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl molybdenum, and alpha-tocopherol; (3) Metal chelating agents Examples include citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid and phosphoric acid.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical composition of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol and polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as, Olive oil and injectable organic esters such as ethyl oleate. The viscosity can be suitably maintained, for example, by using a coating material such as lecithin, or in the case of a dispersion, maintaining the particle size to a required degree, or by using a surfactant.

Such compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Microorganisms can be reliably removed by means of a sterilization process (homologous) and also by including various antibacterial agents and antifungal agents such as parabens, chlorobutanol and phenol sorbic acid. In addition, it may be desirable to include isotonic agents such as sugar and sodium chloride in the composition. In addition, an agent that delays absorption, for example, sodium monostearate and gelatin may be included to prolong the absorption of injectable pharmaceutical formulations.

Pharmaceutically acceptable carriers include sterile powders and sterile dispersions or sterile aqueous solutions for preparing sterile injection solutions or dispersions on the fly. The use of media and preparations for such pharmaceutically active substances is known in the art. Except so far that any conventional medium or agent is incompatible with the active compound, it will be conceivable to use these in the pharmaceutical compositions of the present invention. Supplementary active compounds can also be incorporated into this composition.

The therapeutic composition should be sterilized and stable during manufacture and storage in general. These compositions can be formulated as solutions, microemulsions, liposomes or other structured structures suitable for high concentration drugs. The carrier can be, for example, a dispersion medium or solvent containing water, ethanol, polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.), and suitable mixtures thereof. The viscosity can be suitably maintained, for example, by using a coating, such as lecithin, or maintaining the particle size to a required degree in the case of a dispersion, or by using a surfactant. In many cases, it will be desirable to include isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition. Agents that delay absorption in the composition, such as monostearate salts and gelatin, can be included in the composition to prolong absorption of the injectable composition.

Sterile injectable solutions may be prepared by incorporating one of the above-listed ingredients or a combination of these ingredients, as needed, with the required amount of active compound in a suitable solvent, and then sterile microfiltration. In general, dispersions are prepared from the ingredients listed above by incorporating the active compound into a sterile vehicle (ie, a vehicle containing the basic dispersion medium and other necessary ingredients). In the case of a sterilized powder for preparing a sterile injectable solution, as a preferred manufacturing method, there is a vacuum drying method and a freeze-drying method (freeze drying method) in which a powder composed of an active ingredient and optional additional ingredients can be obtained from a previously sterilized filtered solution.

The amount of active ingredient that can be mixed with the carrier material to produce a single dosage form will depend on the individual to be treated and the specific mode of administration. The amount of active ingredient that can be mixed with the carrier material to form a single dosage form will generally be the amount of the composition that exhibits a therapeutic effect. In general, the amount is 1/100% of the active ingredient, i.e., about 0.01% to about 99%, preferably about 0.1 to about 70%, most preferably about 1 to about, together with a pharmaceutically acceptable carrier. It will be 30%.

The mode of administration can be tailored to provide the optimal desired response (eg, therapeutic response). For example, a single pill may be administered, or this drug may be divided several times over time, or may be administered in an increase or decrease in proportion to the emergency degree of the treatment situation. It is particularly preferred that the parenteral composition is formulated in a unit dosage form for ease of administration and uniformity of administration. Unit dosage form as used herein means a physically separate unit tailored for unit administration to an individual to be treated; Each unit, together with the necessary pharmaceutical carrier, contains a predetermined amount of active compound formulated to produce the desired therapeutic effect. A description of the unit dosage form of the present invention is given, which includes (a) the unique properties of the active compound and the specific therapeutic effect to be achieved, and (b) the synthesis of compounds in the industry, for example, the synthesis of active compounds for the treatment of sensitization in individuals. It is directly influenced by industry-specific limits.

In administering the antibody, the dosage is about 0.0001 to 100 mg, more generally 0.01 to 5 mg per 1 kg of the body weight of the host. For example, the dosage is 0.3 mg/kg of body weight, 1 mg/kg of body weight, 3 mg/kg of body weight, 5 mg/kg of body weight, or 10 mg/kg of body weight or 1-10 mg/kg of body weight. Can be As a typical treatment method, once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or 3 to 6 Includes administration once a month. A preferred mode of administration for the anti-PD-1 antibody of the present invention includes intravenous administration as much as 1 mg per 1 kg of body weight or 3 mg per 1 kg of body weight, wherein the antibody is one of the following administration schedules. It is administered according to: (i) a total of 6 doses every 4 weeks, followed by every 3 months; (ii) administered every 3 weeks; (iii) After 3 mg/kg of body weight was administered once, 1 mg/kg of body weight was administered every 3 weeks.

In some methods, two or more monoclonal antibodies having different binding specificity factors are administered simultaneously, in which case the dosage of each antibody administered is administered so as to fall within the above dosage range. Antibodies are generally administered in a number of cases. The interval between administrations can be, for example, 1 week, 1 month, 3 months or 1 year. The interval may also be non-constant depending on the measurement of the blood level of antibodies to the target antigen in the patient. In some methods, the dosage can be tailored so that the concentration of the antibody in the plasma is about 1-1000 μg/ml, and in some methods it is about 25-300 μg/ml.

Alternatively, the antibody can be administered as a delayed release formulation, in which case the number of administrations should be reduced. The dosage and frequency of administration depend on the half-life of the antibody in the patient's body. In general, the half-life is the longest for human antibodies, followed by humanized, chimeric, and non-human antibodies. The dosage and frequency of administration may vary depending on whether the purpose of treatment is for prophylaxis or treatment. If it is for prophylaxis, it is administered relatively rarely, in relatively small doses over a long period of time. Some patients continue to receive treatment for the rest of their lives. In the case of treatment, relatively large doses are administered with relatively short intervals until the progression of the disease is slowed down or terminated, and preferably until the patient's disease symptoms are partially or completely relieved. Thereafter, the patient can be treated for prophylaxis.

The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may vary in order to obtain the amount of the active ingredient effective to obtain a desired level of therapeutic response to a specific patient, composition and mode of administration without harming the patient. have. The dosage level chosen depends on a variety of pharmacokinetic factors, such as the activity of the particular composition of the invention used, or an ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, Other drugs, compounds and/or substances used with the particular composition employed, the age, sex, weight, condition, overall medical history of the patient to be treated, etc. will depend on factors well known in the medical industry.

The "therapeutically effective amount" of PD-1 of the present invention is to reduce the severity of disease symptoms, increase the number of administrations, and prolong the period in which the symptoms of the disease do not appear, or prevent damage or disorder due to the pain of the disease. For example, in the treatment of tumors, the "therapeutically effective amount" is preferably about 20% or more, more preferably about 40% or more, even more preferably cell growth or tumor growth, compared to an untreated individual. It is intended to mean an inhibitory amount of at least about 60%, and most preferably at least about 80%. The ability of compounds to inhibit tumor growth can be assessed in animal model systems that can predict efficacy against human tumors. Alternatively, this property of the composition can be assessed by observing the inhibitory ability of the compound, which in vitro inhibitory ability can be assessed by assays known to those skilled in the art. The therapeutically effective amount of the therapeutic compound is an amount capable of reducing the size of a tumor or alleviating symptoms in an individual. The person skilled in the art will be able to determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration chosen.

In another aspect, the invention provides a pharmaceutical kit consisting of parts comprising an anti-PD-1 antibody and an anti-CTLA-4 antibody as described herein. This kit may further include instructions for use in the treatment of hyperproliferative diseases (eg, cancer as described herein). In another embodiment, an anti-PD-1 antibody and an anti-CTLA-4 antibody may be included jointly as a unit dosage form.

In certain embodiments, two or more monoclonal antibodies with different binding specificity factors (e.g., anti-PD-1 and anti-CTLA-4) are administered simultaneously, in which case the administration of each antibody administered. The amount falls within the range given above. The antibody may be administered as a single dosage form, or more generally may be administered in multiple divided doses. The interval between single administrations can be, for example, 1 week, 1 month, 3 months or 1 year. The interval may also not be constant as the patient measures blood levels of antibodies to the target antigen. In some methods, the dosage can be tailored so that the concentration of the antibody in the plasma is about 1-1000 μg/ml, and in some methods it is about 25-300 μg/ml.

The compositions of the present invention may be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route of administration and/or mode of administration will depend on the desired outcome. Preferred routes of administration for the antibodies of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion. The phrase “parenteral administration” as used herein refers to a mode of administration excluding intestinal administration and topical administration, generally, an administration method by injection, such as intravenous, intramuscular, intraarterial, Intrasecondary, intracapsular, intraorbital, intracardiac, intradermal, peritoneal, transtracheal, subcutaneous, subcuticle, intraarticular, subcapsular, subarachnoid, intrathecal, epidural and intrasternal injections and injections. , But is not limited thereto.

Alternatively, the antibody of the present invention may be administered by a non-parenteral route, e.g., topical, epithelial or intramucosal route of administration, e.g., intranasal, oral, vaginal, rectal, sublingual or topical administration. It can be administered via the route.

The active compound may be formulated with a carrier that will prevent rapid release of the compound, for example in the form of controlled release formulations such as implants, transdermal patches and microcapsule delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyhydric anhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. A number of methods for preparing such formulations are patented or are generally known to the person skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

The therapeutic composition can be administered with a medical device known in the art. For example, in a preferred embodiment, the therapeutic compound of the present invention is a needleless hypodermic injection device, eg, US Pat. No. 5,399,163; 5,383,851; No. 5,312,335; No. 5,064,413; 4,941,880; 4,790,824; Alternatively, it may be administered through a device disclosed in No. 4,596,556. Examples of well-known implants and dosage forms useful in the present invention include: injectable micro-injection pumps for dispensing pharmaceutical products at controlled rates (US Pat. No. 4,487,603); Jangji for treatment for injecting medicines through the skin (US Patent No. 4,486,194); Pharmaceutical infusion pumps for delivering pharmaceuticals at accurate infusion rates (US Pat. No. 4,447,233); Implantable variable fluid injection device for continuous drug delivery (US Pat. No. 4,447,224); An osmotic drug delivery system with multiple chamber compartments (US Pat. No. 4,439,196); And osmotic drug delivery systems (US Pat. No. 4,475,196). These patents are incorporated herein by reference. A number of other implants, delivery systems and dosage forms are known to those of skill in the art.

In certain embodiments, human monoclonal antibodies of the invention can be formulated as a form that ensures proper distribution in vivo. For example, the blood brain barrier (BBB) excretes a number of highly hydrophilic compounds. (If desired) In order to determine whether the therapeutic compound of the present invention passes through the BBB, the therapeutic compound may be formulated in, for example, liposomes. For methods of making liposomes, see US Patent Nos. 4,522,811; 5,374,548; And 5,399,331. Liposomes may contain one or more moieties that are selectively migrated to specific cells or organs, thereby facilitating targeted drug delivery (see, eg, V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Representative targeting moieties include folic acid or biotin (eg, US Pat. No. 5,416,016 (Low et al.)); Mannoside (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); Antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); Surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); And K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346:123; JJ. Killion; LJ. Fidler (1994J Immunomethods 4:273).

Uses and methods of the present invention

The antibodies, antibody compositions and methods of the present invention have a number of in vitro and in vivo utility including, for example, enhancement of the immune response by blocking action of PD-1, or detection of PD-1. In a preferred embodiment, the antibody of the invention is a human antibody. For example, such molecules may be administered to cells in culture, in vitro or ex vivo, or may be administered in human subjects, such as in vivo, to enhance immunity in various conditions. Therefore, in one aspect, the present invention provides a method of modifying an immune response in a subject, the method comprising administering to the subject the antibody or antigen-binding portion thereof, thereby improving the immune response in the subject. Includes. Preferably, the reaction is enhanced, promoted or up-regulated.

As used herein, the term “individual” is meant to include humans and non-human animals. Non-human animals include all vertebrates such as mammals and non-mammals such as non-human primates, sheep, dogs, cats, cattle, horses, chickens, amphibians and reptiles, for example For example, non-human primates, sheep, mammals such as dogs, cats, cattle and horses are preferred. Preferred individuals include human patients who need to strengthen the immune response. This method is particularly suitable for treating human patients with diseases that can be treated by increasing the T cell mediated immune response. In certain embodiments, the methods of the invention are particularly suitable for treating cancer cells in vivo. To achieve antigen-specific enhancement of immunity, an anti-PD-1 antibody can be administered with an antigen of interest. When antibodies against PD-1 are administered with other agents, the two can be administered sequentially or simultaneously.

The present invention also provides a method of detecting the presence of human PD-1 antigen in a sample, or a method of measuring the amount of human PD-1 antigen, wherein the complex between the antibody or a portion thereof and human PD-1 is Under conditions capable of being formed, a human monoclonal antibody or antigen-binding portion thereof that specifically binds to human PD-1 comprises contacting a sample and a control sample. Thereafter, whether or not the complex is formed is detected. Here, the difference in the degree of complex formation between the samples compared to the control sample is evidence that the human blood antigen is present in the sample.

If the specific binding ability of the antibody against PD-1 of the present invention is compared with CD28, ICOS and CTLA-4, the antibody of the present invention can be used to specifically detect PD-1 expression on the cell surface. In addition, it can also be used to purify PD-1 through an immunoaffinity purification process.

cancer

The blocking action of PD-1 by antibodies can enhance the immune response to cancer cells in patients. The ligand for PD-1, that is, PD-L1, is not expressed in normal human cells, but is expressed in large amounts in many human cancers [Dong et al. (2002) Nat Med 8:787-9]. The interaction between PD-1 and PD-L1 leads to a decrease in tumor-invasive lymphocytes, a decrease in T cell receptor mediated proliferation, and immune evasion by cancer cells [Dong et al. (2003) J Mol Med 81:281- 7; Blank et al. (2005) Cancer Immunol Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100]. Immunosuppression can be reversed by inhibiting the local interaction between PD-1 and PD-L1, and the effect is doubled when the interaction between PD-1 and PD-L2 is blocked [Iwai et al. (2002). PNAS 99: 12293-7; Brown' and others (2003) J. Immunol. 170: 1257-66]. Previous studies have suggested that T cell proliferation can be restored by inhibiting the interaction of PD-1 and PD-L1, whereas blocking the interaction of PD-1/PD-L1 directly affects cancer tumor growth in vivo. There are no reports of this effect as yet. In one aspect, the present invention relates to a method of treating a subject in vivo by inhibiting the growth of a cancer tumor alone using an anti-PD-1 antibody. Anti-PD-1 antibodies can be used to inhibit the growth of cancer tumors. Alternatively, anti-PD-1 antibodies can be used in conjunction with other immunogenic agents, standard cancer therapies or other antibodies, as described below.

Therefore, in one embodiment, the present invention provides a method of inhibiting the growth of tumor cells in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-1 antibody or antigen-binding portion thereof. Includes. Preferably, the antibody is a human anti-PD-1 antibody (eg, any of the human anti-human PD-1 antibodies as described herein). Additionally or alternatively, the antibody may be a chimeric anti-PD-1 antibody or a humanized anti-PD-1 antibody.

Preferred cancers whose growth can be inhibited when the antibody of the present invention is used include cancers that usually respond to immunotherapy. Non-limiting examples of what is preferable as a therapeutic cancer include melanoma (e.g., metastatic malignant melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), breast cancer, Colon cancer and lung cancer (eg, non-small cell lung cancer). Additionally, the subject of treatment of the present invention includes refractory or recurrent malignant tumors whose growth may be inhibited when using the antibody of the present invention.

Examples of other cancers that can be treated using the method of the present invention include bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin and intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer. , Uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, female vulvar carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue Sarcoma, urethral cancer, penile cancer, chronic and acute leukemia, e.g., acute myeloid leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney cancer, ureteral cancer, Renal pelvic carcinoma, central nervous system (CNS) neoplasia, primary CNS lymphoma, tumor neovascularization, spinal axis tumor, brain stem gliocytoma, pituitary adenocarcinoma, Kaposi's sarcoma, epidermal cancer, squamous cell carcinoma, T cell lymphoma , Cancer caused by the environment, for example, cancer caused by asbestos, and combinations of these cancers. The present invention is also useful in the treatment of metastatic cancer, particularly metastatic cancer expressing PD-L1 [Iwai et al. (2005) Int. Immunol. 17:133-144].

Optionally, antibodies to PD-1 are transfected with immunogenic agents such as cancer cells, purified tumor antigens (e.g., recombinant proteins, peptides and carbohydrate molecules), cells and genes encoding immune-promoting cytokines. Can be administered with cells [He et al. (2004) J. Immunol. 173:4919-28]. Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, e.g., gp100, MAGE antigens, Trp-2, MART1 and/or peptides of tyrosinase, or tumor cells transfected to express the cytokine GM-CSF. (Described in detail below).

In humans, some tumors are thought to be immunogenic, such as melanoma. It is expected that by raising the threshold of T cell activation by blocking PD-1, tumor responses in the host can be activated.

PD-1 blocking action appears to be most effective when combined with the vaccination process. A number of experimental techniques have been devised for vaccination against tumors [Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C, 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; And Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043, DeVita, V. et al. (eds.), 1997, Cancer: Principles and Practice of Oncology. See Fifth Edition]. In one of these techniques, vaccines are prepared using autologous tumor cells or allogeneic tumor cells. Such a cellular vaccine is considered to be most effective when tumor cells are transduced to express GM-CSF. GM-CSF is known to be a potent active factor in providing antigens for tumor vaccination [Dranoff et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90: 3539-43].

As a result of studying gene expression and gene expression patterns on a large scale in various tumors, it was possible to define a so-called tumor-specific antigen [Rosenberg, SA (1999) Immunity 10: 281-7]. In many cases, such tumor-specific antigens include differentiation antigens expressed in tumors and cells from which the tumor is derived, such as melanocyte antigens gp100, MAGE antigen and Trp-2. More importantly, the majority of these antigens can be viewed as targets of tumor-specific T cells found in the host. In order to elicit an immune response against these proteins, PD-1 blocking action can be utilized with a group of recombinant proteins and/or peptides expressed in tumors. These proteins are usually regarded as their antigens by the immune system and thus become resistant to these self-antigens. Tumor antigens may also contain protein telomerase (a protein necessary for the synthesis of telomeres of chromosomes, expressed at 85% or more in human cancer, and expressed only at a limited level in somatic tissues) [Kim, N et al. (1994). Science 266: 2011-2013]. (These somatic tissues can be protected from immune attack by a variety of methods.) Tumor antigens are also fusion proteins between two unrelated sequences (i.e., bcr-abl of the Philadelphia chromosome), or sinus derived from B cell tumors. It may also be a "neo-antigen" expressed in cancer cells due to somatic mutations that produce a reference specimen type or modify protein sequences.

Other tumor vaccines may include proteins derived from viruses involved in human cancer, such as human papilloma virus (HPV), hepatitis viruses (HBV and HCV) and Kaposi herpes sarcoma virus (KHSV). Another form of tumor-specific antigen that can be utilized with PD-1 blocking action is a purified heat shock protein (HSP) isolated from the tumor tissue itself. Such heat shock proteins contain fragments of proteins derived from tumor cells, and these HSPs are very effectively delivered to antigen-providing cells for inducing tumor immunity [Suot, R & Srivastava, P (1995) Sciene 269: 1585-1588; Tamura, Y. et al. (1997) Science 278:117-120].

Dendritic cells (DC) are potent antigen presenting cells that can be used to prime antigen-specific responses. DCs are produced extracellularly and can be loaded with various protein and peptide antigens and tumor cell extracts [Nestle, F. et al. (1998) Nature Medicine 4: 328-332]. DCs can also be transduced by genetic means that express such tumor antigens. DCs are also fused directly to tumor cells for immunization [Kugler, A. et al. (2000) Nature Medicine 6:332-336]. As one method of vaccination, DC immunization can be performed efficiently in conjunction with PD-1 blockade, thereby activating the anti-tumor response more strongly.

PD-1 blockade can also be performed in conjunction with standard cancer treatments. PD-1 blockade can be performed effectively in conjunction with chemotherapy regimens. In such cases, the dose of chemotherapy reagent administered may be reduced [Mokyr, M. et al. (1998) Cancer Research 58: 5301-5304]. As an example of such a combination method, there is a method of administering dicarbazine and an anti-PD-1 antibody for the treatment of melanoma. Another example of such a combination method is a method of administering interleukin-2 (IL-2) and an anti-PD-1 antibody together for the treatment of melanoma. The scientific theory on which the combination of PD-1 blockade and chemotherapy is based is that apoptosis, i.e., as a result of the cytotoxic action of most chemotherapy compounds, increases the level of tumor antigens in the antigen-providing pathway. I can do it. Other combination therapies that can exhibit a synergistic effect by blocking PD-1 through cell death include radiation treatment, surgery, and hormone blocking. Each of these methods forms a source of tumor antigens in the host. Angiogenesis inhibitors can be administered in addition to blocking PD-1. Inhibition of neovascularization leads to a process of tumor cell death that can supply tumor antigens to the host antigen presentation pathway.

PD-1 blocking antibodies may also be used with bispecific antibodies that target Fc alpha or Fc gamma receptor-expressing effector cells to tumor cells (see, eg, US Pat. Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. For example, anti-Fc receptor/anti-tumor antigen (eg, Her-2/neu) bispecific antibodies are used to target macrophages to tumor sites. Such targeting can more efficiently activate tumor specific responses. In this response, the T cell arm will increase by PD-1 blocking action. Alternatively, antigens can be delivered directly to DCs via bispecific antibodies that bind to tumor antigens and dendritic cell specific cell surface markers.

Tumors evade the host's immune surveillance function by a variety of mechanisms. Many of these mechanisms can be overcome by inactivating immunosuppressive proteins expressed by the tumor. Examples of such proteins include TGF-beta [Kehrl, J. et al. (1986) J. Exp. Med. 163: 1037-1050], IL-10 [Howard, M. & O'Garra, A. (1992) Immunology Today 13: 198-200], and Fas ligand [Hahne, M. et al. (1996) Science 274: 1363-1365]. Antibodies to each of these proteins can be used in conjunction with anti-PD-1, resulting in interfering with the efficacy of the immunosuppressant and promoting the tumor immune response by the host.

Other antibodies that can be used to activate host immune responsiveness can be used with anti-PD-1. Such antibodies include molecules on the surface of dendritic cells that activate DC and antigen-presenting functions. Anti-CD40 antibodies can effectively replace T cell aiding activity [Ridge, J. et al. (1998) Nature 393: 474-478], and can be used together with PD-1 antibodies [Ito, N. et al. 2000) Immunobiology 201 (5) 527-40]. T cell co-stimulatory molecules such as CTLA-4 (e.g., U.S. Patent No. 5,811,097), OX-40 (Weinberg, A. et al. (2000) Immunol 164: 2160-2169), 4-1BB (Melero , I et al. (1997) Nature Medicine 3: 682-685 (1997)), and ICOS (Hutloff, A. et al. (1999) Nature 397: 262-266). You can also increase it.

In order to treat various tumors of hematopoietic origin, bone marrow transplantation is currently being used. As a result of such treatment, graft-to-host disease occurs, but treatment benefits may be obtained from the graft-to-tumor response. PD-1 blockade can be used to increase the efficacy of donors transplanted into tumor specific T cells.

In addition, for antigen-specific T cells for tumors, several experimental treatment methods, including the steps of extracellular activation and proliferation of antigen-specific T cells, and selective migration of these cells to receptors, are available. It also exists [Greenberg, R. & Riddell, S. (1999) Science 285: 546-51]. These methods can also be used to activate T cell responses to infectious agents, such as CMV. Extracellular activation occurring in the presence of an anti-PD-1 antibody is expected to increase the number and activity of selectively migrated T cells.

Infectious disease

Other methods of the present invention are used to treat patients exposed to certain toxins or pathogens. Therefore, another aspect of the present invention provides a method of treating an infectious disease in a subject, wherein the subject can be treated for the infectious disease by administering an anti-PD-1 antibody or antigen-binding portion thereof to the subject. Includes the steps to make. Preferably, the antibody is a human anti-human PD-1 antibody (eg, any of the human anti-PD-1 antibodies as described herein). Additionally or alternatively, the antibody may be a chimeric antibody or a humanized antibody.

As described above, similar to when this antibody is administered to a tumor, antibody-mediated PD-1 blockade can be utilized alone, as an adjuvant, or in combination with a vaccine, resulting in pathogens, toxins and self- It can promote an immune response to an antigen. Examples of pathogens from which a particularly useful effect can be expected by such a treatment method include pathogens for which there is currently no effective vaccine, or pathogens for which conventional vaccines do not show full effect. Examples include HIV virus, hepatitis virus (A, B, & C), influenza virus, herpes virus, Giardia, malaria, Leishmania, Staphylococcus aureus , Pseudomonas aureus. Including, but not limited to, Pseudomonas Aeruginosa. PD-1 blockade is particularly useful for infections established by agents such as HIV that present modified antigens during infection. Since such a novel epitope is recognized as foreign upon administration of anti-human PD-1, it induces a strong T cell response that is not attenuated by a negative signal through PD-1.

Some examples of pathogenic viruses causing infection treatable by the method of the present invention include HIV, hepatitis (type A, B or C), Herpes virus (e.g., VZV, HSV-1, HAV-6, HSV -II and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, ecovirus, rhino virus, coxsackie virus , Cornovirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia Vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus encephalitis virus).

Some examples of infection-causing pathogenic bacteria treatable by the method of the present invention include Chlamydia, Rickettsia bacteria, Mycobacteria, Staphylococcus, Streptococcus, Pneumonococcus, meningococcus and conococcus, Klebsi. Klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacillus, cholera, titanus ), botulism, anthrax, plague, leptospirosis and Lymes disease bacteria.

Some examples of pathogenic fungi treatable cause infection by the method of the present invention, Candida (Candida) (albicans (albicans), krusei (krusei), glabrata (glabrata), trophy faecalis (tropicalis), etc.), Cryptococcus Lactococcus Neo Fort scanned only (Cryptococcus neoformans), Aspergillus (Aspergillus) (Fu fumigatus (fumigatus), Niger (niger), etc.), non-Corrales in (Temuco (mucor), Absecon Dia (absidia), Rizzo Perth (rhizophus )), Sporothrix schenkii , Blastomyces dermatitidis , Paracoccidioides brasiliensis , Coccidioides immitis and histo Plasma capsulatum (Histoplasma capsulatum ).

Some examples of infection-causing pathogenic parasites treatable by the method of the present invention include Entamoeba histolytica , Balantidium coli , Naegleriafowleri , and Acanthamoeba species. ( Acanthamoeba sp. ), Giardia lambi a, Cryptosporidium sp. , Pneumocystis carinii , Plasmodium vivax , and Babesia microti ( Babesia microti ), Trypanosoma brucei , Trypanosoma cruzi , Leishmania donovani , Toxoplasma gondi , and Nippostrongylus brasiliensis . do.

In all of the foregoing methods, PD-1 blockade is a different form of immunotherapy, e.g., cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2), or dual specific antibody therapy. (Increases the amount of tumor antigen presentation) [eg, Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2:1121-1123].

Autoimmune reaction

Anti-PD-1 antibodies can elicit and amplify an autoimmune response. Indeed, by inducing anti-tumor responses using tumor cells and peptide vaccines, many anti-tumor responses are anti-autoreactive [anti-CTLA-4+ GM-CSF-modified B16 melanoma observed in pigment loss (Elsas And many others (same as above)); Depigmentation in Trp-2 vaccinated mice (Overwijk, W. et al. (1999) Proc. Natl. Acad Sci. USA 96: 2982-2987]; Autoimmune prostatecosis induced by TRAMP tumor cell vaccine (Hurwitz, A. (2000) homolog), melanoma peptide antigen vaccination and skin pigment loss (vitilago) observed in human clinical trials (Rosenberg, SA and White, DE (1996) J. Immunother Emphasis Tumor Immunol 19. (1): 81-4] was involved.

Therefore, in order to devise a vaccination method that effectively induces an immune response against such a magnetic protein for disease treatment, a method of blocking anti-PD-1 while administering various magnetic proteins can be considered. For example, Alzheimer's disease involves inadequate accumulation of Aβ peptides in amyloid deposits inside the brain; Antibody reactions to this amyloid can eliminate these amyloid deposits [Schenk etal, (1999) Nature 400: 173-177].

Other magnetic proteins can also be used as targets for the treatment of allergies and asthma, such as IgE, and targets for the treatment of rheumatoid arthritis, such as TNFα. Finally, antibody responses to various hormones can be induced by using anti-PD-1 antibodies. Neutralizing antibody responses to reproductive hormones can be used in contraception. Neutralizing antibodies to hormones and other soluble factors required for the growth of certain tumors may also be considered possible vaccination targets.

Methods analogous to the above-described methods using anti-PD-1 antibodies include inappropriate accumulations of other self-antigens such as amyloid deposits such as Aβ of Alzheimer's disease, cytokines such as TNFα, and IgE. It can be used to induce a therapeutic autoimmune response to treat patients with

vaccine

The anti-PD-1 antibody can be used to promote an antigen-specific immune response by administering an anti-PD-1 antibody and an antigen of interest (eg, a vaccine) together. Therefore, in another aspect of the invention, the invention provides a method of enhancing an immune response to an antigen in a subject, the method comprising: (i) an antigen; And (ii) administering the anti-PD-1 antibody or antigen-binding portion thereof, thereby enhancing the immune response to the antigen in the subject. Preferably, the antibody is a human anti-human PD-1 antibody (eg, any of the human anti-PD-1 antibodies as described herein). Additionally or alternatively, the antibody may be a chimeric antibody or a humanized antibody. The antigen can be, for example, a tumor antigen, a viral antigen, a bacterial antigen or an antigen derived from a pathogen. Non-limiting examples of such antigens include the antigens described in the section above, such as tumor antigens (or tumor vaccines) as described above, or antigens derived from viruses, bacteria or other pathogens as described above.

The antibody composition of the present invention (e.g., human monoclonal antibodies, multispecific molecules and bispecific molecules and immunoconjugates) are well known in the art for suitable routes of administration for in vivo and in vitro administration. , It can be selected by the person concerned. For example, the antibody composition can be administered by injection (eg, intravenous injection or subcutaneous injection). The appropriate dosage of the molecule used will depend on the age and weight of the individual and the concentration and/or formulation of the antibody composition.

As already mentioned above, the human anti-PD-1 antibodies of the invention can be administered in combination with one or more other therapeutic agents, such as cytotoxic agents, radiotoxic agents or immunosuppressants. The antibody may be administered in combination with the agent (as an immune complex), or may be administered separately from the agent. In the latter case (separate administration), the antibody may be administered before, after or concurrently with the administration of the agent, or may be administered in conjunction with other known therapies such as anticancer therapy such as radiotherapy. Such therapeutic agents include anti-neoplastic agents such as doxorubicin (adiamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil, dicarbazine and cyclophosphamide hydroxyurea, which are toxic to patients. It is only valid when used at a level indicating or less than that. Cisplatin is administered intravenously at 100 mg per dose once every 4 weeks, and adriamycin is administered intravenously at 60 to 75 mg per dose once every 21 days. When the human anti-PD-1 antibody or antigen-binding fragment thereof of the present invention is administered together with a chemotherapy agent, two anticancer agents that act through different mechanisms exhibiting cytotoxic effects on human tumor cells are provided. Such co-administration can solve the problem of changing the antigenicity of the tumor cells, which makes the tumor cells not responding to the antibody, or the problem of developing resistance to drugs.

Kits comprising the antibody composition of the present invention (eg, human antibody, bispecific or multispecific molecule or immunoconjugate) and instructions for use are also included within the scope of the present invention. The kit further comprises one or more additional reagents, or one or more additional human antibodies according to the present invention (e.g., a human antibody having a complementary activity that binds to an epitope of the PD-1 antigen distinct from the first human antibody). Can include. Kits typically include a label indicating how to use the contents of the kit. Here, the term "label" means including any recorded or recorded material that is supplied on or separately from the kit, or attached to the kit.

Combination therapy

The present invention is based in part on the following experimental data. The mouse tumor model (MC38 colon cancer and SA1/N fibrosarcoma) was used to observe the efficacy of tumor treatment in vivo by administering the therapeutic immunostimulating antibody-anti-CTLA-4 and anti-PD-1 together. Immunocombination therapy was performed concurrently with implantation of tumor cells (Examples 14 and 17), or for a time sufficient for tumor to settle after tumor cells were implanted (Examples 15, 16 and 18). Regardless of the timing of antibody treatment, only anti-CTLA-4 antibody treatment, and only anti-PD-1 antibody (chimeric antibody in which rat anti-mouse PD-1 was modified with mouse immunoglobulin Fc region, see Example 1) When this was done, it was found that tumor growth was slightly reduced in the MC38 tumor model (see Figs. 21, 24 and 27). Treatment with anti-CTLA-4 antibody alone was quite efficient in the SA1/N tumor model (see Fig. 30D), but in parallel studies through this model, the dose of anti-CTLA-4 antibody had to be reduced more than usual. did. However, when the anti-CTLA-4 antibody and the anti-PD-1 antibody were treated together, it was found that tumor growth was significantly reduced compared to when only one of these two antibodies was treated (Fig. 21D. , 24d, 30f and 33h-33j). In addition, the results of Examples 14, 16 and 18, when treated with an anti-CTLA-4 antibody and an anti-PD-1 antibody, compared to when only one of these two antibodies was treated, the optimal treatment administration It was found that even if administered in an amount less than the amount, it had a significant (synergistic) effect on tumor growth (ie, when the dose was administered at a dose less than the therapeutic dose, the combination therapy was more effective than that of a single therapy. Was more efficient]. Without being bound by any theory, by raising the threshold of T cell activation by PD-1 and CTLA-4 blockade, anti-tumor responses in the host can be activated.

In one embodiment, the invention provides a method of treating a hyperproliferative disease, the method comprising administering to the subject a PD-1 antibody and a CTLA-4 antibody. In a further embodiment, the anti-PD-1 antibody is administered at a subtherapeutic dose, the anti-CTLA-4 antibody is administered at a subtherapeutic dose, or both antibodies are It is administered at a dosage less than or equal to the therapeutic dose. In another embodiment, the present invention provides a method for altering the event of an opposite action associated with the treatment of a hyperproliferative disease using an immune stimulating agent, the method comprising an anti-PD-1 antibody and a sub-therapeutic dose of anti -Administering the CTLA-4 antibody to the subject. In certain embodiments, the subject is a human. In certain embodiments, the anti-CTLA-4 antibody is a human sequence monoclonal antibody 10D1, and the anti-PD-1 antibody is a human sequence monoclonal antibody such as 17D8, 2D3, 4H1, 5C4 and 4A11. have. Human sequence monoclonal antibodies 17D8, 2D3, 4H1, 5C4 and 4A11 were isolated and structurally characterized as disclosed in US Provisional Patent Application No. 60/679,466.

Anti-CTLA-4 antibody and anti-PD-1 monoclonal antibody (mAb) and human sequence antibody of the present invention are used in various techniques, such as conventional monoclonal antibody methods, for example, standard somatic cell hybridization techniques ( Kohler and Milstein (1975) Nature 256:495). Any technique for producing monoclonal antibodies can be used, such as viral transformation of B lymphocytes or oncogenic transformation. One animal system for producing hybridomas is the murine system. The method of producing hybridomas in mice is a well-established method. Techniques and immunization methods for isolating immunized splenocytes for fusion are known in the art. Fusion partners (eg, murine myeloma cells) and methods of fusion are also known (see, eg, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York. ].

The anti-CTLA-4 antibody of the present invention binds to an epitope present on human CTLA-4 and can inhibit this CTLA-4 from interacting with the human B7 counterpart receptor. Since the interaction of human CTLA-4 with human B7 transmits a signal that leads to the inactivation of T cells bearing the human CTLA-4 receptor, the antagonism of this interaction is the result of the antagonism of the human CTLA-4 receptor-bearing T cells. It effectively induces the activation of and increases its amount or prolongs the period, and as a result, the duration of the immune response is also extended, and the degree thereof is also amplified. The US patent for anti-CTLA-4 antibody 5,811,097; 5,855,887; 6,051,227; PCT application publications WO 01/14424 and WO 00/37504; And disclosed in US Patent Publication 2002/0039581. Each of these references is specifically incorporated herein by reference to describe anti-CTLA-4 antibodies. Representative clinical anti-CTLA-4 antibodies are human monoclonal antibody 10D1 as disclosed in WO 01/14424 and US Patent Application No. 09/644,668. Antibody 10D1 alone or in combination with vaccine, chemotherapy, or interleukin-2, in single and multiple doses, in more than 500 patients i.e. metastatic melanoma, prostate cancer, lymphoma, renal cell carcinoma, breast cancer, ovarian cancer And to a patient diagnosed with HIV. Other anti-CTLA-4 antibodies to be included in the method of the present invention include, for example, WO 98/42752; WO 00/37504; US Patent No. 6,207,156; Hurwitz et al. (1998) Proc. Natl. Acad. Sci. USA 95(17): 10067-10071; Camacho et al. (2004) J. Clin. Oncology 22(145): Abstract No. 2505 (antibody CP-675206); And Mokyr et al. (1998) Cancer Res. 58:5301-5304]. In certain embodiments, the method of the invention comprises using a human sequence antibody, preferably an anti-CTLA-4 antibody, which is a monoclonal antibody, and in other embodiments comprises using a monoclonal antibody 10D1. .

In certain embodiments, the anti-CTLA-4 antibody _{binds human CTLA-4 with a K D} 5×10 ^-8 M or less, or with human CTLA-4 with a K _D 1×10 ^-8 M or less, or CTLA-4 and K _D 5 × 10 ^-8 M or less, or human CTLA-4 and K _D 1 × 10 ^-8 M or less and 1 × 10 ^-10 M or less.

In order to enhance the immune response against hyperproliferative diseases by blocking PD-1 and CTLA-4, it is useful to use a combination of antibodies. In a preferred embodiment, the antibody of the invention is a human antibody. For example, these molecules can be administered to cells in culture, in vitro or ex vivo, or can be administered to a human subject, for example, in vivo, resulting in the ability to enhance immunity in various states. . Therefore, in one aspect, the present invention provides a method of modifying an immune response in a subject, wherein the method is administered to a subject in combination with the antibody of the present invention, or in combination with the antigen-binding portion of the antibody, Modifying the immune response in the subject. Preferably, the reaction is enhanced, promoted or up-regulated. In another embodiment, the present application provides a method of altering the event of an opposite action induced in treating a hyperproliferative disease with an immunostimulating therapeutic agent, the method comprising an anti-PD-1 antibody and a sub-therapeutic dose. Administering an anti-CTLA-4 antibody to the subject.

Blocking PD-1 and CTLA-4 by antibodies can enhance the patient's immune response to cancer cells. Cancers whose growth is inhibited when the antibody of the present invention is used generally include cancers that are responsive to immunotherapy. Examples of cancers that respond to treatment by the combination therapy of the present invention include melanoma (eg, metastatic malignant melanoma), kidney cancer, prostate cancer, breast cancer, colon cancer, and lung cancer. Examples of other cancers that can be treated when using the method of the present invention include bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin and eye malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, Testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, female vulvar carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, Soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, e.g., acute myeloid leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney cancer, ureteral cancer , Renal pelvic carcinoma, central nervous system (CNS) neoplasia, primary CNS lymphoma, tumor neovascularization, spinal axis tumor, brain stem gliocytoma, pituitary adenocarcinoma, Kaposi's sarcoma, epidermal cancer, squamous cell carcinoma, T cell Lymphoma, cancer caused by the environment, such as cancer caused by asbestos, and combinations of these cancers. The present invention is also useful in the treatment of metastatic cancer.

In certain embodiments, the therapeutic antibodies disclosed herein may be combined and administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions each containing an antibody in a pharmaceutically acceptable carrier. I can. In other embodiments, therapeutic antibodies may be administered in combination and sequentially. For example, anti-CTLA-4 antibody and anti-PD-1 antibody may be administered sequentially, for example, anti-CTLA-4 antibody is administered first, followed by anti-PD-1 antibody, or , Anti-PD-1 antibody can be administered first, followed by anti-CTLA-4 antibody. In addition, if the parallel therapy is performed one or more times sequentially, the order may be reversed during continuous administration, or the antibodies may be administered by maintaining the same order but at each administration time point, and continuous administration may be performed at the same time or at the same time. It can be performed in any combination. For example, first anti-CTLA-4 antibody and anti-PD-1 antibody can be administered simultaneously, and then, anti-CTLA-4 → anti-PD-1 can be sequentially administered, and further Finally, it can be administered continuously in the order of anti-PD-1 → anti-CTLA-4. Other representative dosing regimens may include the first administration [anti-PD-1 → anti-CTLA-4] and subsequent administration.

Optionally, when administered in combination with an anti-PD-1 antibody and an anti-CTLA-4 antibody, also immunogenic agents such as cancer cells, purified tumor antigens (e.g., recombinant proteins, peptides and carbohydrate molecules) , Cells, and cells transfected with genes encoding immune-promoting cytokines [He et al. (2004) J. Immunol. 173:4919-28]. Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, e.g., gp100, MAGE antigens, Trp-2, MART1 and/or peptides of tyrosinase, or tumor cells transfected to express the cytokine GM-CSF. (Described in detail below).

The joint blocking action of PD-1 and CTLA-4 can be performed in conjunction with the vaccination process. A number of experimental techniques have been devised for vaccination against tumors [Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C, 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; And Restifo and Sznol, Cancer Vaccines, Ch. 61, pp. 3023-3043, DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology. See Fifth Edition]. In one of these techniques, vaccines are prepared using autologous tumor cells or allogeneic tumor cells. Such a cellular vaccine is considered to be most effective when tumor cells are transduced to express GM-CSF. GM-CSF is known to be an effective active factor when providing antigens for tumor vaccination [Dranoff et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90: 3539-43].

As a result of studying gene expression and gene expression patterns on a large scale in various tumors, it was possible to define a so-called tumor-specific antigen [Rosenberg (1999) Immunity 10: 281-7]. In many cases, such tumor-specific antigens include differentiated antibodies expressed in tumors and cells from which the tumors are derived, such as melanocyte antigens gp100, MAGE antigen, and Trp-2. More importantly, the majority of these antigens are identified as targets of tumor-specific T cells found in the host. In certain embodiments, to elicit an immune response against such a protein, the process of blocking PD-1 and CTLA-4 together using an antibody composition disclosed herein is performed with a group of recombinant proteins and/or peptides expressed in the tumor. Can be used together. These proteins usually regard the immune system as their antigens and are therefore resistant to these self-antigens. Tumor antigens may also contain protein telomerase (a protein necessary for the synthesis of telomeres of chromosomes, expressed by 85% or more in human cancer, and expressed only at a limited level in somatic tissue) [Kim et al. (1994) Science 266. : 2011-2013]. (These somatic tissues can be protected from immune attack by a variety of methods.) Tumor antigens are also fusion proteins between two unrelated sequences (i.e., bcr-abl of the Philadelphia chromosome), or sinus derived from B cell tumors. It may also be a "neo-antigen" expressed in cancer cells due to somatic mutations that produce a reference specimen type or modify protein sequences.

Other tumor vaccines may include proteins derived from viruses involved in human cancer, such as human papilloma virus (HPV), hepatitis viruses (HBV and HCV) and Kaposi herpes sarcoma virus (KHSV). Another form of tumor-specific antigen that can be utilized with PD-1 blockade is purified heat shock protein (HSP) isolated from the tumor tissue itself. Such heat shock proteins contain fragments of proteins derived from tumor cells, and these HSPs are delivered very effectively to antigen-providing cells to induce tumor immunity [Sout & Srivastava (1995) Science 269:1585-1588; Tamura et al. (1997) Science 278:117-120].

Dendritic cells (DC) are potent antigen presenting cells that can be used to prime antigen-specific responses. DCs are produced extracellularly and can be loaded with various protein and peptide antigens and tumor cell extracts [Nestle et al. (1998) Nature Medicine 4: 328-332]. DCs can also be transduced by genetic means that express such tumor antigens. DCs are also fused directly to tumor cells for immunization [Kugler et al. (2000) Nature Medicine 6:332-336]. As one method of vaccination, DC immunization can be performed efficiently in conjunction with co-blockade of PD-1 and CTLA-4, thereby activating the anti-tumor response more potently.

Co-blockade of PD-1 and CTLA-4 can also be performed in conjunction with standard cancer therapy. For example, co-blockade of PD-1 and CTLA-4 can be performed in efficient association with chemotherapy regimens. In this case, the dosage of any chemotherapy reagent administered together with the antibody of the present invention may be reduced as well as when the anti-PD-1 antibody and the anti-CTLA-4 antibody are administered together [Mokyr, M. et al. (1998) Cancer Research 58: 5301-5304]. An example of such a combination method is a method of administering dicarbazine, an anti-PD-1 antibody, and an anti-CTLA-4 antibody for the treatment of melanoma. Another example of such a combination method is a method of administering an anti-PD-1 antibody and an anti-CTLA-4 antibody together with interleukin-2 (IL-2) for the treatment of melanoma. The scientific theory on which the combination of PD-1 and CTLA-4 blockade and chemotherapy is based is a result of apoptosis, i.e., the cytotoxic action of most chemotherapy compounds, in the antigen-providing pathway. Is that it can increase the level of. Other combination therapies that can exhibit a synergistic effect by blocking PD-1 and CTLA-4 together through cell death include radiation treatment, surgery, and hormone blocking. Each of these methods forms a source of tumor antigens in the host. Angiogenesis inhibitory factors can be implemented in conjunction with co-blockade of PD-1 and CTLA-4. The inhibition of neovascularization leads to the death of tumor cells that can supply the tumor antigen to the host antigen presentation pathway.

PD-1 blocking antibodies and CTLA-4 blocking antibodies may also be used with bispecific antibodies targeting Fc alpha or Fc gamma receptor-expressing effector cells to tumor cells (see, e.g., U.S. Pat. 5,837,243]. Bispecific antibodies can be used to target two separate antigens. For example, anti-Fc receptor/anti-tumor antigen (eg, Her-2/neu) bispecific antibodies are used to target macrophages to tumor sites. Such targeting can more efficiently activate tumor specific responses. The T cell arm of this response will be increased by the co-blocking action of PD-1 and CTLA-4. Alternatively, antigens can be delivered directly to DCs via bispecific antibodies that bind to tumor antigens and dendritic cell specific cell surface markers.

In another embodiment, an anti-PD-1 antibody and an anti-CTLA-4 antibody are combined with an anti-neoplastic antibody such as Rituxan® (rituximab; rituximab), Herceptin® (trast Main map; trastuzumab), Bexxar® (tositumomab), Zevalin® (ibritumomab), Camppath® (alemtuzumab), Limposide ( Lymphocide)® (eprtuzumab; eprtuzumab), Avastin® (bevacizumab) and Tarceva® (erlotinib), and the like. Without being bound by any theory, for example, anti-cancer antibodies or anti-cancer antibodies conjugated to toxins that enhance the immune response mediated by CTLA-4 or PD-1 are cancer cells (e.g., tumor cells). Can induce the death of In a representative embodiment, as a method of treating hyperproliferative diseases (e.g., cancer, tumor), anti-PD-1 and anti-CTLA-4 antibodies, which can enhance an anti-tumor immune response by a host, are used as anti-cancer antibodies. It may include a method of administering simultaneously or sequentially, or optionally in combination with each other.

Tumors evade the host's immune surveillance function by a variety of mechanisms. Many of these mechanisms are expressed by tumors and can be overcome by inactivation of immunosuppressive proteins. Examples of such proteins include TGF-β (Kehrl, J. et al. (1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard, M. & O'Garra, A. (1992) Immunology Today 13: 198-200), and Fas ligand (Hahne, M. et al. (1996) Science 274: 1363-1365). In other embodiments, antibodies against each of these entities can also be administered in combination with anti-PD-1 and anti-CTLA-4, resulting in neutralizing the efficacy of the immunosuppressant and anti-tumor immune response by the host. Can promote.

Other antibodies that can be used to activate the host's immune responsiveness can also be used with anti-PD-1 and anti-CTLA-4. Such antibodies include molecules present on the surface of dendritic cells that activate DC and antigen-presenting functions. Anti-CD40 antibodies can effectively replace T cell auxotroph activity [Ridge, J. et al. (1998) Nature 393: 474-478], and can be used with anti-PD-1 antibodies and anti-CTLA-4 antibodies. [Ito, N. et al. (2000) Immunobiology 201 (5) 527-40]. T cell co-stimulatory molecules such as OX-40 (Weinberg, A. et al. (2000) Immunol 164: 2160-2169), 4-1BB (Melero, I et al. (1997) Nature Medicine 3: 682-685 ( 1997), and ICOS (Hutloff, A. et al. (1999) Nature 397: 262-266) can also increase the level of T cell activation.

In order to treat various tumors of hematopoietic origin, bone marrow transplantation is currently being used. As a result of such treatment, graft-to-host disease occurs, but treatment benefits may be obtained from the graft-to-tumor response. Co-blockade of PD-1 and CTLA-4 can be used to increase the efficacy of donors implanted in tumor specific T cells.

In addition, for antigen-specific T cells for tumors, several experimental treatment methods, including the steps of extracellular activation and proliferation of antigen-specific T cells, and selective migration of these cells to receptors, are available. It also exists [Greenberg, R. & Riddell, S. (1999) Science 285: 546-51]. These methods can also be used to activate T cell responses to infectious agents, such as CMV. Extracellular activation occurring in the presence of anti-PD-1 antibody and anti-CTLA-4 antibody is expected to increase the number and activity of selectively migrated T cells.

As presented herein, organs, e.g., the GI tract and skin after treatment with an anti-CTLA-4 antibody, are subject to immune-promoting therapeutic antibody therapy and events of immune-related counteraction [e.g., GI tract ( Diarrhea and enteritis) and skin (fever and pruritus)]. For example, after treatment with anti-CTLA-4 antibodies, events of non-colonal gastrointestinal immunity-related adverse action can occur in the esophagus (esophagitis), duodenum (duodenitis) and ileum (enteritis).

In certain embodiments, the present invention provides a method of altering the event of an opposite action induced upon treatment of a hyperproliferative disease with an immune stimulating agent, the method comprising providing an anti-PD-1 antibody and a sub-therapeutic dose to the individual. And administering an anti-CTLA-4 antibody of. For example, the methods of the invention provide a method of reducing the incidence of immunostimulating therapeutic antibody-induced colitis or diarrhea by administering a non-absorbable steroid to a patient. Since any patient to be administered an immunostimulatory therapeutic antibody is at risk of developing colitis or diarrhea caused by this antibody, the entire patient population is suitable for treatment according to the method according to the present invention. Although steroids were administered to treat inflammatory bowel disease (IBD) and prevent the worsening of IBD, this steroid was not used to prevent (reduce the incidence) of IBD in patients who were not diagnosed with IBD. . When using steroids (non-absorbable steroids), an event of serious opposite action has discouraged the desire to use it for prophylaxis.

In a further embodiment, the co-blocking effect of PD-1 and CTLA-4 (i.e., immunostimulating therapeutic antibody with anti-PD-1 and anti-CTLA-4) is achieved when any non-absorbable steroid is used together. It can be doubled. As used herein, "non-absorbable steroid" is a glucocorticoid that initially undergoes a wide range of transit mechanisms, and is a steroid whose biocompatibility is lowered to less than about 20% after the mechanism in the liver progresses. In one embodiment of the invention, the non-absorbable steroid is budesonide. Budesonide is a topically-acting glucocorticoid that is widely metabolized mainly by the liver when administered orally. ENTOCORTEC® (Astra-Zeneca) is a pH-dependent and time-dependent oral dosage form of budesonide developed to optimize drug delivery through the colon to the ileum. This Entocortek® is approved in the United States for the treatment of mild to moderate Crohn's disease (associated with the bowel and/or upper colon). When treating Crohn's disease, the oral dosage of Entocortek® is typically 6-9 mg/day. This Entocortec® is released into the small intestine before being absorbed, where it resides in the intestinal mucosa. Once it passes through the intestinal mucosa target tissue, this Entocortek® is extensively metabolized by the cytochrome P450 system in the liver to metabolites with trace amounts of glucocorticoid activity. Therefore, the biocompatibility is low (about 10%). Due to the low biocompatibility of budenosides, the cure rate is improved compared to other glucocorticoids with less extensive first-pass metabolism. Budenoside reduces events of opposite action, such as hypothalamic-pituitary inhibitory action, compared to systemic-acting corticosteroids. However, long-term administration of this Entocortek® may lead to systemic glucocorticoid effects such as hypercortisolosis and adrenaline suppression. See PDR 58th ed. 2004; 608-610].

In another embodiment, when the co-blocking effect of PD-1 and CTLA-4 (i.e., immunostimulating therapeutic antibodies anti-PD-1 and anti-CTLA-4) is administered together with a non-absorbable steroid, salicylate is also Can be administered together. As salicylate, 5-ASA preparations such as sulfasalazine (AZULFIDINE®, Pharmacia &UpJohn); Olsalazine (DIPENTUM®, Pharmacia &UpJohn); Valsalazide (COLAZAL®, Salix Pharmaceuticals, Inc.); And mesalazide (ASACOL®, Procter & Gamble Pharmaceuticals; PENTASA®, Shire US; CANASA®, Axcan Scandipharm, Inc.; ROWASA®, Solvay). Includes.

According to the method of the present invention, anti-PD-1 and anti-CTLA-4 antibodies, and salicylate administered in combination with a non-absorbable steroid, are used to reduce the incidence of colitis induced by immunostimulating antibodies. It may be administered simultaneously or sequentially with the absorbable steroid. Therefore, for example, a method for reducing the incidence of colitis induced by an immunostimulating antibody, according to the present invention, comprises the steps of administering salicylate and a non-absorbable steroid simultaneously or sequentially (e.g., non-absorbable And administering salicylate 6 hours after administering the steroid), or administering these substances in any combination. In addition, according to the present invention, the salicylate and the non-absorbable steroid can be administered by the same route (e.g., both can be administered by the oral route of administration), or can be administered by different routes. (E.g., salicylate may be administered orally, non-absorbable steroids may be administered rectally), in this case, the route(s) used to administer the anti-PD-1 antibody and anti-CTLA-4 antibody. ) Can be different.

The invention may be described in more detail by the following examples which should not be construed as further limiting the invention. The references, patents, and published patent applications and drawings cited in their entirety are incorporated herein by reference.

1A shows the amino acid sequence (SEQ ID NO: 1) and the nucleotide sequence (SEQ ID NO: 57) of the heavy chain variable region of the 17D8 human monoclonal antibody. CDR1 (SEQ ID NO: 15), CDR2 (SEQ ID NO: 22) and CDR3 (SEQ ID NO: 29) regions are underlined, and V, D and J germline derivatives are also indicated.
1B shows the amino acid sequence (SEQ ID NO: 8) and the nucleotide sequence (SEQ ID NO: 64) of the light chain variable region of the 17D8 human monoclonal antibody. CDR1 (SEQ ID NO: 36), CDR2 (SEQ ID NO: 43) and CDR3 (SEQ ID NO: 50) regions are underlined, and V and J germline derivatives are also indicated.
2A shows the amino acid sequence (SEQ ID NO: 2) and the nucleotide sequence (SEQ ID NO: 58) of the heavy chain variable region of a 2D3 human monoclonal antibody. CDR1 (SEQ ID NO: 16), CDR2 (SEQ ID NO: 23) and CDR3 (SEQ ID NO: 30) regions are underlined, and V and J germline derivatives are also indicated.
2B shows the amino acid sequence (SEQ ID NO: 9) and the nucleotide sequence (SEQ ID NO: 65) of the light chain variable region of a 2D3 human monoclonal antibody. CDR1 (SEQ ID NO: 37), CDR2 (SEQ ID NO: 44) and CDR3 (SEQ ID NO: 51) regions are underlined, and V and J germline derivatives are also indicated.
3A shows the amino acid sequence (SEQ ID NO: 3) and the nucleotide sequence (SEQ ID NO: 59) of the heavy chain variable region of a 4H1 human monoclonal antibody. CDR1 (SEQ ID NO: 17), CDR2 (SEQ ID NO: 24) and CDR3 (SEQ ID NO: 31) regions are underlined, and V and J germline derivatives are also indicated.
3B shows the amino acid sequence (SEQ ID NO: 10) and the nucleotide sequence (SEQ ID NO: 66) of the light chain variable region of the 4H1 human monoclonal antibody. CDR1 (SEQ ID NO: 38), CDR2 (SEQ ID NO: 45) and CDR3 (SEQ ID NO: 52) regions are underlined, and V and J germline derivatives are also indicated.
4A shows the amino acid sequence (SEQ ID NO: 4) and the nucleotide sequence (SEQ ID NO: 60) of the heavy chain variable region of a 5C4 human monoclonal antibody. CDR1 (SEQ ID NO: 18), CDR2 (SEQ ID NO: 25) and CDR3 (SEQ ID NO: 32) regions are underlined, and V and J germline derivatives are also indicated.
4B shows the amino acid sequence (SEQ ID NO: 11) and the nucleotide sequence (SEQ ID NO: 67) of the light chain variable region of the 5C4 human monoclonal antibody. CDR1 (SEQ ID NO: 39), CDR2 (SEQ ID NO: 46) and CDR3 (SEQ ID NO: 53) regions are underlined, and V and J germline derivatives are also indicated.
5A shows the amino acid sequence (SEQ ID NO: 5) and the nucleotide sequence (SEQ ID NO: 61) of the heavy chain variable region of the 4A11 human monoclonal antibody. CDR1 (SEQ ID NO: 19), CDR2 (SEQ ID NO: 26) and CDR3 (SEQ ID NO: 33) regions are underlined, and V and J germline derivatives are also indicated.
5B shows the amino acid sequence (SEQ ID NO: 12) and the nucleotide sequence (SEQ ID NO: 68) of the light chain variable region of the 4A11 human monoclonal antibody. CDR1 (SEQ ID NO: 40), CDR2 (SEQ ID NO: 47) and CDR3 (SEQ ID NO: 54) regions are underlined, and V and J germline derivatives are also indicated.
6A shows the amino acid sequence (SEQ ID NO: 6) and the nucleotide sequence (SEQ ID NO: 62) of the heavy chain variable region of the 7D3 human monoclonal antibody. CDR1 (SEQ ID NO: 20), CDR2 (SEQ ID NO: 27) and CDR3 (SEQ ID NO: 34) regions are underlined, and V and J germline derivatives are also indicated.
6B shows the amino acid sequence (SEQ ID NO: 13) and the nucleotide sequence (SEQ ID NO: 69) of the light chain variable region of the 7D3 human monoclonal antibody. CDR1 (SEQ ID NO: 41), CDR2 (SEQ ID NO: 48) and CDR3 (SEQ ID NO: 55) regions are underlined, and V and J germline derivatives are also indicated.
7A shows the amino acid sequence (SEQ ID NO: 7) and the nucleotide sequence (SEQ ID NO: 63) of the heavy chain variable region of the 5F4 human monoclonal antibody. CDR1 (SEQ ID NO: 21), CDR2 (SEQ ID NO: 28) and CDR3 (SEQ ID NO: 35) regions are underlined, and V and J germline derivatives are also indicated.
7B shows the amino acid sequence (SEQ ID NO: 14) and the nucleotide sequence (SEQ ID NO: 70) of the light chain variable region of the 5F4 human monoclonal antibody. CDR1 (SEQ ID NO: 42), CDR2 (SEQ ID NO: 49) and CDR3 (SEQ ID NO: 56) regions are underlined, and V and J germline derivatives are also indicated.
Fig. 8 shows the arrangement of the amino acid sequence of the heavy chain variable region of 17D8, 2D3, 4H1, 5C4 and 7D3 and the amino acid sequence of human germline V _H 3-33 (SEQ ID NO: 71).
Fig. 9 shows the arrangement of the amino acid sequence of the light chain variable region of 17D8, 2D3 and 7D3 and the human germline V _κ L6 amino acid sequence (SEQ ID NO: 73).
Fig. 10 shows the arrangement of the amino acid sequence of the light chain variable region of 4H1 and 5C4 and the human germline V _κ L6 amino acid sequence (SEQ ID NO: 73).
Fig. 11 shows the arrangement of the amino acid sequence of the heavy chain variable region of 4A11 and 5F4 and the human germline V _H 4-39 amino acid sequence (SEQ ID NO: 72).
Fig. 12 shows the arrangement of the amino acid sequence of the light chain variable region of 4A11 and 5F4 and the human germline V _κ L15 amino acid sequence (SEQ ID NO: 74).
13A-13B show the results of flow cytometry experiments demonstrating that 5C4 and 4H1, which are human monoclonal antibodies generated against human PD-1, bind to the cell surface of CHO cells transfected with full-length human PD-1. To indicate. 13A shows a flow cytometry graph for 5C4. 13B shows a graph of flow cytometry for 4H1. The thin line indicates binding to CHO cells, and the thick line indicates binding to CHO hPD-1 cells.
14 is a graph demonstrating that the human monoclonal antibodies 17D8, 2D3, 4H1, 5C4 and 4A11 produced against human PD-1 specifically bind to PD-1 and do not bind to other members of the CD28 group. Is to represent.
15A-15C show the results of a flow cytometry experiment demonstrating that human monoclonal antibodies 4H1 and 5C4, which are human monoclonal antibodies generated against human PD-1, bind to PD-1 on the cell surface. 15A shows that the antibody binds to activated human T cells. 15B shows that the antibody binds to cynomolgus monkey T cells. 15C shows that the antibody binds to CHO transfected cells expressing PD-1.
16A-16C show the results of experiments demonstrating that human monoclonal antibodies against human PD-1 promote T cell proliferation, IFN-gamma secretion, and IL-2 secretion in a mixed lymphocyte response assay. 16A is a bar graph showing concentration dependent T cell proliferation; 16B is a bar graph showing concentration dependent IFN-gamma secretion; 16C is a bar graph showing concentration-dependent IL-2 secretion.
17A-17B are flow cytometry demonstrating that a human monoclonal antibody against human PD-1 blocks the binding of PD-L1 and PD-L2 to CHO-transfected cells expressing PD-1. It shows the results of the experiment. 17A is a graph showing that the antibody inhibits the binding of PD-L1; 17B is a graph showing that the antibody inhibits the binding of PD-L2.
Fig. 18 shows the results of a flow cytometry experiment showing that a human monoclonal antibody against human PD-1 does not promote apoptosis of T cells.
19 is an experiment demonstrating that the effect of anti-PD-1 HuMab on IFN-gamma secretion by PBMCs derived from CMV-positive donors is concentration dependent when PBMC are promoted with CMV lysate and anti-PD-1. It represents the result of.
FIG. 20 shows the results of tumor growth experiments in a mouse model system, demonstrating that in vivo treatment of a mouse tumor with an anti-PD-1 antibody inhibits the growth of this tumor.
21A to 21D show the tumor volume over time in each mouse implanted with MC38 colon tumor cells (PD-L1 ⁻ ), and the results when treated with any of the following treatments on the same day: ( A) mouse IgG (control), (B) anti-CTLA-4 antibody, (C) anti-PD-1 antibody, and (D) anti-CTLA-4 antibody and anti-PD-1 antibody. As described in Example 13, changes in tumor volume after treatment with antibodies to mice on the 3rd, 6th, and 10th days after the start of the experiment were observed over 60 days.
Figure 22 shows the average tumor volume of the mouse shown in Figure 21.
23 shows the median tumor volume of the mouse shown in FIG. 21.
24A to 24D show the tumor volume over time in each mouse implanted with MC38 colon tumor cells (PD-L1 ⁻ ) and the results after 1 week after treatment with the following treatment: ( A) mouse IgG (control), (B) anti-CTLA-4 antibody, (C) anti-PD-1 antibody and (D) anti-CTLA-4 antibody and anti-PD-1 antibody. The tumor volume at the first day after treatment was about 315 mm 3. As described in Example 14, the antibodies were administered to the mice at the lapse of Day 3, Day 6 and Day 10.
Figure 25 shows the average tumor volume of the mice shown in Figure 24.
26 shows the median tumor volume of the mouse shown in FIG. 24.
Figure 27 shows the mean (day 7) of tumor volumes over time in each mouse implanted with MC38 colon tumor cells (PD-L1 ^{-), and day 0 after transplantation (as described in Example 15),} It shows the result of treatment with any of the following treatments at the elapse of days 3, 6 and 10: (A) Mouse IgG (control) (20 mg/kg, X ₂₀ ) (B) Anti-PD-1 antibody (10 mg/kg) and mouse IgG (10 mg/kg) (P ₁₀ X ₁₀ ), (C) anti-CTLA-4 antibody (10 mg/kg) and mouse IgG (10 mg/kg) kg) (C ₁₀ X ₁₀ ), (D) anti-CTLA-4 antibody and anti-PD-1 antibody (each 10 mg/kg) (C ₁₀ P ₁₀ ), (E) anti-CTLA-4 antibody and anti -PD-1 antibody (3 mg/kg each) (C ₃ P ₃ ), and (F) anti-CTLA-4 antibody and anti-PD-1 antibody (1 mg/kg each) (C ₁ P ₁ ). Two of the mouse groups were sequentially treated with the following antibodies, respectively: (G) anti-CTLA-4 antibody (10 mg/kg, day 0), anti-CTLA-4 antibody (10 mg/kg, Day 3), anti-PD-1 antibody (10 mg/kg, day 6), and anti-PD-1 antibody (10 mg/kg, day 10) (C ₁₀ C ₁₀ P ₁₀ P ₁₀ ); And (H) anti-PD-1 antibody (10 mg/kg, day 0), anti-PD-1 antibody (10 mg/kg, day 3), anti-CTLA-4 antibody (10 mg/kg, Day 6), and anti-CTLA-4 antibody (10 mg/kg, day 10) (10 mg/kg, day 10) (P ₁₀ P ₁₀ C ₁₀ C ₁₀ ).
Figure 28 shows the average tumor volume of the mice shown in Figure 27.
FIG. 29 shows the median value of the tumor volume of the mouse shown in FIG. 27.
30A to 30F show the tumor volume over time in each mouse implanted with SA1/N fibrosarcoma cells (PD-L1 ⁻ ) and the first day after treatment with any of the following therapeutic agents. It shows tumor volume: (A) PBS (vehicle control), (B) mouse IgG (antibody control, 10 mg/kg), (C) anti-PD-1 antibody (10 mg/kg), (D) anti -CTLA-4 antibody (10 mg/kg), (E) anti-CTLA-4 antibody (0.2 mg/kg), (F) anti-PD-1 antibody (10 mg/kg) and anti-CTLA-4 antibody (0.2 mg/kg). As described in Example 16, on the 4th, 7th, and 11th days after the initiation of the experiment, the volume of the tumor after treatment with the antibody in mice was observed over 41 days.
Figure 31 shows the average tumor volume of the mice shown in Figure 29.
Fig. 32 shows the median value of the tumor volume of the mouse shown in Fig. 29.
FIG 33a~ 33j are also SA1 / N fibrosarcoma cells (PD-L1 ^-) over time in tumor volume in each of the mice transplanted with and, after transplantation 7th, 10th day, 13th day and 17th il It represents the tumor volume after treatment with any of the following therapeutic agents at elapsed time (as described in Example 17): (A) PBS (vehicle control), (B) mouse IgG (antibody control, 10 mg/) kg), (C) anti-CTLA-4 antibody (0.25 mg/kg), (D) anti-CTLA-4 antibody (0.5 mg/kg), (E) anti-CTLA-4 antibody (5 mg/kg) , (F) anti-PD-1 antibody (3 mg/kg), (G) anti-PD-1 antibody (10 mg/kg), (H) anti-PD-1 antibody (10 mg/kg) and anti -CTLA-4 antibody (0.25 mg/kg), (I) anti-PD-1 antibody (10 mg/kg) and anti-CTLA-4 antibody (0.5 mg/kg), and (F) anti-PD-1 Antibody (3 mg/kg) and anti-CTLA-4 antibody (0.5 mg/kg). On the first day after treatment, the tumor volume was about 110 mm 3.
Figure 34 shows the average tumor volume of the mice shown in Figure 33.
Figure 35 shows the median value of the tumor volume of the mouse shown in Figure 33.
36A and 36B show tumor volumes over time in each mouse transplanted with SA1/N fibrosarcoma cells (PD-L1 ⁻ ) and days 10, 13, 16 and 19 after transplantation. It represents the tumor volume after treatment with any of the following therapeutic agents at elapsed time (as described in Example 17): (A) mouse IgG (antibody control, 10 mg/kg) or (B) anti-PD -1 antibody (10 mg/kg) and anti-CTLA-4 antibody (1 mg/kg). On the first day of treatment, the tumor volume was about 250 mm 3.
Figure 37 shows the average tumor volume of the mice shown in Figure 36.
FIG. 38 shows the median tumor volume of the mouse of FIG. 36.
39 shows the average and median values of tumor suppression rates calculated from the tumor volumes shown in FIGS. 33 and 36.
40A to 40B show tumor volumes in BALB/c mice injected with RENCA renal adenocarcinoma cells (PD-L1 ⁺ ) subcutaneously [Murphy and Hrushesky (1973) J. Natl. Cancer Res. 50:1013-1025] (when the 12th day has elapsed) and when one of the following therapeutic agents is injected into the peritoneum after the 0th, 3rd, 6th, and 9th days after the injection Results are shown: (A) mouse IgG (antibody control, 20 mg/kg), (B) anti-PD-1 antibody (10 mg/kg), (C) anti-CTLA-4 antibody (10 mg/kg) ), and (D) co-administration of anti-PD-1 antibody (10 mg/kg) and anti-CTLA-4 antibody (10 mg/kg). On the first day of treatment, the tumor volume was about 115 mm 3.
Figure 41 shows that the binding of mouse PD-L2-Fc fusion protein and mouse PD-1 (mPD-1) is blocked by anti-mPD-1 antibody 4H2 in a dose dependent manner. Binding is detected by measuring the fluorescence of FITC-labeled donkey-anti-rat IgG by ELISA. The greater the MFI (average fluorescence intensity), the greater the degree of binding.
Figure 42 shows the binding curve of the anti-mPD-1 antibody to the immobilized mPD-1-Fc fusion protein through ELISA.
43 shows the binding curve of the rat anti-mPD-1 antibody 4H2.B3 to mPD-1-expressing CHO cells. The binding was detected using FITC conjugated donkey-anti-rat IgG, and the result was measured by FACS (MFI).
Figure 44 shows the binding curve of mPD-1-expressing CHO cells and mPD-L1-hFc fusion protein when the concentration of anti-mPD-1 antibody 4H2.B3 is increased. The binding was detected using FITC conjugated goat-anti-human IgG, and the result was measured by FACS (MFI).
Figure 45 shows the binding curve of the anti-mPD-1 antibody 4H2.B3 and mPD-1-expressing CHO cells when compared with the case of the chimeric rat: mouse anti-mPD-1 antibody 4H2.
Figure 46 is the binding curve of mPD-1-expressing CHO cells and mPD-L1-hFc fusion protein when increasing the concentration of rat anti-mPD-1 antibody 4H2.B3 or chimeric rat: mouse anti-mPD-1 antibody 4H2 Is to represent.
Indicates the mean tumor volume of the re-inoculated with a tumor-free ^mice Figure 47 is pre-treated with -PD-1 antibody, wherein SA1 / N fibrosarcoma cells (PD-L1). The average tumor volume of the original mice (control, pre-inoculated or untreated mice) transplanted with SA1/N fibrosarcoma cells (PD-L1 ^{−) was also shown.}
48 is MC38 colon tumor cells (PD-L1 ^-) processes the transplant after the treatment hayeotgeona wherein -PD-1 antibody, or wherein the -PD-1 antibody and anti--CTLA-4 antibody with, and more when the first treatment It shows the change in tumor volume over time in tumor-free mice that survived when re-implanted with a 10-fold greater amount of MC38 crystal tumor cells. The average tumor volume of the original mice (control, pre-inoculated or untreated mice) transplanted with MC38 colon tumor cells was also shown.
Figure 49 shows the average tumor volume of the mice shown in Figure 48.
50 shows the average tumor volume over time in each mouse transplanted with CT26 colon tumor cells.
51A to 51B show experimental results demonstrating that a human monoclonal antibody against human PD-1 promotes T cell proliferation and IFN-gamma secretion in a culture medium containing T regulatory cells. 50A is a bar graph showing the results of concentration-dependent T cell proliferation using HuMAb 5C4; 50B is a bar graph showing the results of concentration-dependent IFN-gamma secretion using HuMAb 5C4.
52A-52B show experimental results demonstrating that a human monoclonal antibody against human PD-1 promotes T cell proliferation and IFN-gamma secretion in a culture medium containing activated T cells. 51A is a bar graph showing the results of concentration-dependent T cell proliferation using HuMAb 5C4; 51B is a bar graph showing the results of concentration-dependent IFN-gamma secretion using HuMAb 5C4.
Figure 53 shows antibody dependent cytotoxicity (ADCC) demonstrating that human monoclonal anti-PD-1 antibody kills human activated T cells in an ADCC concentration-dependent manner with respect to the Fc region of the anti-PD-1 antibody. ) It shows the result of the test.
Figure 54 shows the results of a complement dependent cytotoxicity (CDC) assay demonstrating that human monoclonal anti-PD-1 antibodies do not kill human activated T cells in a CDC concentration-dependent manner.

Example 1: Generation of human monoclonal antibody against PD-1

antigen

Immunization method using both antigens, i.e., (i) a recombinant fusion protein containing the extracellular portion of PD-1, and (ii) a membrane-bound full-length PD-1 as antigens. The two antigens were produced by recombinant transfection methods in CHO cell lines.

Transgenic HuMab and KM Mouse™

Using the HCo7 variant of the HuMab transgenic mouse and the KM variant of the transgenic transchromosomal mouse (each expressing a human antibody gene), a monoclonal antibody (antibody against PD-1) that is entirely human was prepared. I did. In each of the above mouse strains, the endogenous mouse kappa light chain gene was disrupted in a recessive isotype [Chen et al. (1993) EMBO J. 12:811-820], and the endogenous mouse heavy chain gene was implemented in PCT publication WO 01/09187. It was destroyed in a recessive isomorphic state as described in Example 1. Each of these mouse strains carries the human kappa light chain transgene, KCo5 [Fishwild et al. (1996) Nature Biotechnology 14:845-851]. The HCo7 variant carries the HCo7 human heavy chain transgene [US Pat. No. 5,545,806; 5,625,825; And 5,545,807]. The KM variant carries the SC20 trans chromosome [PCT publication WO 02/43478].

HuMab and KM immunization

In order to produce a monoclonal antibody (antibody against PD-1) that is entirely human, HuMab mice and KM mice™ were used with purified recombinant PD-1 fusion protein and PD-1-transfected CHO cells (antigen ). For general immunization procedures for HuMab mice, see Lonberg, N. et al. (1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851 and PCT Publication WO 98/24884. The age of the mice when the antigen was first injected was 6 to 16 weeks of age. Immunization by intraperitoneal, subcutaneous (Sc) injection or leg injection of HuMab mice and KM mice™ using purified recombinant preparations (5-50㎍) of PD-1 fusion protein antigen and 5-10×10 ^{6 cells.} Made it.

Transgenic mice were intraperitoneally injected with antigens from either complete Freund's adjuvant or Ribi's adjuvant, and then again with incomplete Freund's or Libi's adjuvant antigens for 3 to 21 days (total 11 Immunized twice or less), and this mouse was immunized twice. Immune response was observed by blood sampling in the posterior orbit. Plasma was screened by ELISA (described below), and mice administered with a sufficient adequate amount of anti-PD-1 human immunoglobulin were used for fusion. After intravenous injection of an antigen into the mouse to strengthen immunity, the mouse was euthanized and the spleen was removed after the third day. Typically, 10 to 35 fusions were performed for each antigen. Dozens of mice were immunized for each antigen.

Selection of HuMab Mice or KM Mice™ Producing Anti-PD-1 Antibodies

To select HuMab mice and KM mice™ producing antibodies that bind to PD-1, sera from immunized mice were tested by ELISA as described in Fishwild, D. et al. (1996). . Briefly, a microtiter plate was coated with recombinant PD-1 fusion protein purified from transformed CHO cells (1-2 μg/ml in PBS) (100 μl/well) and incubated overnight at 4° C. Then, blocking was performed with 200 μl/well of 5% fetal bovine serum (0.05%) in PBS/Tween. Serum dilutions obtained from PD-1-immunized mice were added to each well and incubated for 1-2 hours at room temperature. The plate was washed with PBS/Tween and then incubated for 1 hour at room temperature with a goat-anti-human IgG polyclonal antibody conjugated with horseradish peroxidase (HRP). After washing, the plate was developed with ABTS substrate (Sigma, A-1888, 0.22 mg/ml) and analyzed by spectrometry at OD 415-495. Mice with the highest anti-PD-1 antibody titer were used for fusion. Fusion was performed as described below, and hybridoma supernatants were tested for anti-PD-1 activity by ELISA.

Preparation of hybridoma producing human monoclonal antibody against PD-1

By using PEG based on a standard protocol, or by an electric field electrofusion method using a Cyto Pulse large-chamber cell fusion electric machine (Cyto Pulse Sciences, Inc., Glen Burnie, MD), Mouse spleen cells isolated from HuMAb or KM mice were fused to mouse myeloma cells. Thereafter, the resulting hybridomas were screened for production of antigen-specific antibodies. A single cell suspension of splenocytes obtained from immunized mice was fused with 50% PEG (Sigma) to 1/4 of SP 2/0 non-secreting mouse myeloma cells (ATCC, CRL 1581). The cells were plated on a flat-bottom microtiter plate at a density of about 1×10 ⁵ , and then 10% bovine clone serum, 10% P388D1 (ATCC, CRL TIB-63) conditioning medium, DEME (Mediatech, CRL 10013; high concentration). Glucose, including L-glutamine and sodium pyruvate) in 3 to 5% Origen (IGEN) + 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 mg/ml gentamicin and 1×HAT (Sigma, CRL P-7185) Incubated for 2 weeks in a selective medium containing. After about 1 to 2 weeks, the cells were cultured in a medium in which HAT was changed to HT. Each well was then screened by ELISA for human anti-PD-1 monoclonal IgG antibody. Once excessive hybridoma growth occurred, the medium was generally observed after 10-14 days. Antibody secreting hybridomas were resprayed and screened, and if still positive for human IgG, this monoclonal antibody was subcloned at least twice by limiting dilution. Thereafter, stable subclones were cultured in vitro to produce a small amount of antibodies in tissue culture medium and characterized.

Hybridoma clones 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 were selected for further analysis.

Example 2: Structural characterization of human monoclonal antibodies 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4

Using standard PCR techniques, cDNA sequences encoding the heavy and light chain variable regions of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 monoclonal antibodies were respectively obtained from 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4. After obtaining from hybridomas, these sequences were sequenced using standard DNA sequencing techniques.

The nucleotide and amino acid sequences of the heavy chain variable region of 17D8 are shown in Fig. 1A, SEQ ID NO: 57, and SEQ ID NO: 1, respectively.

The nucleotide and amino acid sequences of the light chain variable region of 17D8 are shown in Fig. 1A, SEQ ID NO: 64, and SEQ ID NO: 8, respectively.

As a result of comparing the 17D8 heavy chain immunoglobulin sequence with the known human germline immunoglobulin heavy chain sequence, the 17D8 heavy chain is a V _{H segment derived from human germline V H} 3-33, an uncrystallized D segment, and human germ line JH4b. It has been demonstrated that the JH segment derived from is used. The result of arranging the 17D8 V _H sequence to the germ line V _H 3-33 sequence is shown in FIG. 8. _{As a result of further analyzing the 17D8 V H} sequence using the Cabot system for determining the CDR regions, as shown in Figs. 1A and 8 and SEQ ID NOs: 15, 22 and 29, respectively, the CDR1, CDR2 and CD3 regions of the heavy chain were derived. I was able to make it.

As a result of comparing the 17D8 light chain immunoglobulin sequence with the known human germline immunoglobulin light chain sequence, the 17D8 light chain uses a V _{L segment derived from human germline V κ} L6 and a JK segment derived from human germ line JK4. Has been proven. The result of arranging the 17D8 V _L sequence to the germline V _κ L6 sequence is shown in FIG. 9. _{As a result of further analysis of the 17D8 V L} sequence using the Cabot system for determining the CDR regions, as shown in FIGS. 1B and 9 and SEQ ID NOs: 36, 43 and 50, respectively, CDR1, CDR2 and CD3 regions of the light chain were derived I was able to make it.

The nucleotide and amino acid sequences of the heavy chain variable portion of 2D3 are shown in Fig. 2A, SEQ ID NO: 58, and SEQ ID NO: 2, respectively.

The nucleotide and amino acid sequences of the light chain variable region of 2D3 are shown in Fig. 2B, SEQ ID NO: 65, and SEQ ID NO: 9, respectively.

As a result of comparing the 2D3 heavy chain immunoglobulin sequence with a known human germline immunoglobulin heavy chain sequence, the 2D3 heavy chain is a V _{H segment derived from human germline V H} 3-33, a D segment derived from human germ line 7-27. , And the use of a JH segment derived from the human germline JH4b has been demonstrated. The result of arranging the 2D3 V _H sequence to the germ line V _H 3-33 sequence is shown in FIG. 8. _{As a result of further analysis of the 2D3 V H} sequence using the Cabot system for determining the CDR regions, as shown in FIGS. 2A and 8 and SEQ ID NOs 16, 23 and 30, respectively, the CDR1, CDR2 and CD3 regions of the heavy chain were derived. I was able to make it.

As a result of comparing the 2D3 light chain immunoglobulin sequence with the known human germline immunoglobulin light chain sequence, the 2D3 light chain uses a V _{L segment derived from human germline V κ} L6 and a JK segment derived from human germ line JK4. Has been proven. The result of arranging the 2D3 V _L sequence to the germ line V _κ L6 sequence is shown in FIG. 9. _{As a result of further analysis of the 2D3 V L} sequence using the Cabot system for determining the CDR regions, as shown in FIGS. 2B and 9 and SEQ ID NOs: 37, 44 and 51, respectively, the CDR1, CDR2 and CDR3 regions of the light chain were derived. I was able to make it.

The nucleotide and amino acid sequences of the heavy chain variable region of 4H1 are shown in Fig. 3A, SEQ ID NO: 59, and SEQ ID NO: 3, respectively.

The nucleotide and amino acid sequences of the light chain variable region of 4H1 are shown in Fig. 3B, SEQ ID NO: 66, and SEQ ID NO: 10, respectively.

As a result of comparing the 4H1 heavy chain immunoglobulin sequence with a known human germline immunoglobulin heavy chain sequence, the 4H1 heavy chain is a V _{H segment derived from human germline V H} 3-33, an uncrystallized D segment, and human germ line JH4b It has been demonstrated that the JH segment derived from is used. The results of arranging the 4H1 V _H sequence with respect to the germ line V _H 3-33 sequence are shown in FIG. 8. _{As a result of further analysis of the 4H1 V H} sequence using the Cabot system for determining the CDR regions, as shown in FIGS. 3A and 8 and SEQ ID NOs: 17, 24 and 31, respectively, the CDR1, CDR2 and CD3 regions of the heavy chain were derived. I was able to make it.

As a result of comparing the 4H1 light chain immunoglobulin sequence with a known human germline immunoglobulin light chain sequence, the 4H1 light chain uses a V _{L segment derived from human germline V κ} L6 and a JK segment derived from human germ line JK1. Has been proven. The results of arranging the 4H1 V _L sequence with respect to the germ line V _κ L6 sequence are shown in FIG. 10. _{As a result of further analysis of the 4H1 V L} sequence using the Cabot system for determining the CDR regions, as shown in FIGS. 3B and 10 and SEQ ID NOs: 38, 45 and 52, respectively, the CDR1, CDR2 and CD3 regions of the light chain were derived. I was able to make it.

The nucleotide and amino acid sequences of the heavy chain variable region of 5C4 are shown in Fig. 4A, SEQ ID NO: 60, and SEQ ID NO: 4, respectively.

The nucleotide and amino acid sequences of the light chain variable region of 5C4 are shown in Fig. 4B, SEQ ID NO: 67, and SEQ ID NO: 11, respectively.

As a result of comparing the 5C4 heavy chain immunoglobulin sequence with a known human germline immunoglobulin heavy chain sequence, the 5C4 heavy chain is a V _{H segment derived from human germline V H} 3-33, an uncrystallized D segment, and human germ line JH4b. It has been demonstrated that the JH segment derived from is used. The result of arranging the 5C4 V _H sequence to the germ line V _H 3-33 sequence is shown in FIG. 8. _{As a result of further analysis of the 5C4 V H} sequence using the Cabot system for determining the CDR regions, as shown in FIGS. 4A and 8 and SEQ ID NOs: 18, 25 and 32, respectively, the CDR1, CDR2 and CD3 regions of the heavy chain were derived. I was able to make it.

As a result of comparing the 5C4 light chain immunoglobulin sequence with a known human germline immunoglobulin light chain sequence, the 5C4 light chain uses a V _{L segment derived from human germline V κ} L6 and a JK segment derived from human germline JK1. Has been proven. The result of arranging the 5C4 V _L sequence with respect to the germ line V _κ L6 sequence is shown in FIG. 10. _{As a result of further analysis of the 5C4 V L} sequence using the Cabot system for determining the CDR regions, as shown in FIGS. 4B and 10 and SEQ ID NOs: 39, 46 and 53, respectively, the CDR1, CDR2 and CD3 regions of the light chain were derived I was able to make it.

The nucleotide and amino acid sequences of the heavy chain variable region of 4A11 are shown in Fig. 5A, SEQ ID NO: 61, and SEQ ID NO: 5, respectively.

The nucleotide and amino acid sequences of the light chain variable region of 4A11 are shown in Fig. 5B, SEQ ID NO: 68, and SEQ ID NO: 12, respectively.

As a result of comparing the 4A11 heavy chain immunoglobulin sequence with a known human germline immunoglobulin heavy chain sequence, the 4A11 heavy chain is a V _{H segment derived from human germline V H} 4-39, a D segment derived from human germ line 3-9. , And the use of a JH segment derived from the human germline JH4b has been demonstrated. The results of arranging the 4A11 V _H sequence for the germline V _H 4-39 sequence are shown in FIG. 11. _{As a result of further analyzing the 4A11 V H} sequence using the Cabot system for determining the CDR region, as shown in FIGS. 5A and 11 and SEQ ID NOs 19, 26 and 33, respectively, the CDR1, CDR2 and CD3 regions of the heavy chain were derived. I was able to make it.

As a result of comparing the 4A11 light chain immunoglobulin sequence with a known human germline immunoglobulin light chain sequence, the 4A11 light chain uses a V _{L segment derived from human germline V κ} L15 and a JK segment derived from human germline JK1. Has been proven. Fig. 12 shows the results of arranging the 4A11 V _L sequence with respect to the germline V _{κ L6 sequence. As a result of further analyzing the 4A11 V L} sequence using the Cabot system for determining the CDR regions, as shown in Figs. 5B and 12 and SEQ ID NOs: 40, 47 and 54, respectively, the CDR1, CDR2 and CD3 regions of the light chain were derived. I was able to make it.

The nucleotide and amino acid sequences of the heavy chain variable region of 7D3 are shown in Fig. 7A, SEQ ID NO: 62, and SEQ ID NO: 6, respectively.

The nucleotide and amino acid sequences of the light chain variable region of 7D3 are shown in Fig. 7B, SEQ ID NO: 69, and SEQ ID NO: 13, respectively.

As a result of comparing the 7D3 heavy chain immunoglobulin sequence with the known human germline immunoglobulin heavy chain sequence, the 7D3 heavy chain is a V _{H segment derived from human germline V H} 3-33, a human germline 7-27 D segment, and a human It has been demonstrated that it uses a JH segment derived from the germline JH4b. Figure 8 shows the results of arranging the 7D3 V _H sequence with respect to the germ line V _{H 3-33 sequence. As a result of further analyzing the 7D3 V H} sequence using the Cabot system for determining the CDR regions, as shown in Figs. 6A and 8 and SEQ ID NOs: 20, 27 and 34, respectively, the CDR1, CDR2 and CD3 regions of the heavy chain were derived. I was able to make it.

As a result of comparing the 7D3 light chain immunoglobulin sequence with the known human germline immunoglobulin light chain sequence, the 7D3 light chain uses a V _{L segment derived from human germline V κ} L6 and a JK segment derived from human germline JK4. Has been proven. Fig. 9 shows the results of arranging the 7D3 V _L sequence with respect to the germ line V _{κ L6 sequence. As a result of further analyzing the 7D3 V L} sequence using the Cabot system for determining the CDR regions, as shown in Figs. 6B and 9 and SEQ ID NOs: 41, 48 and 55, respectively, the CDR1, CDR2 and CD3 regions of the light chain were derived. I was able to make it.

The nucleotide and amino acid sequences of the heavy chain variable region of 5F4 are shown in Fig. 7A, SEQ ID NO: 63, and SEQ ID NO: 7, respectively.

The nucleotide and amino acid sequences of the light chain variable region of 5F4 are shown in Fig. 7B, SEQ ID NO: 70, and SEQ ID NO: 14, respectively.

As a result of comparing the 5F4 heavy chain immunoglobulin sequence with a known human germline immunoglobulin heavy chain sequence, the 5F4 heavy chain is a V _{H segment derived from human germline V H} 4-39, a D segment derived from human germ line 3-9. , And the use of a JH segment derived from the human germline JH4b has been demonstrated. The result of arranging the 5F4 V _H sequence to the germ line V _H 4-39 sequence is shown in FIG. 11. _{As a result of further analyzing the 5F4 V H} sequence using the Cabot system for determining the CDR regions, as shown in Figs. 7A and 11 and SEQ ID NOs: 21, 28 and 35, respectively, the CDR1, CDR2 and CD3 regions of the heavy chain were derived. I was able to make it.

As a result of comparing the 5F4 light chain immunoglobulin sequence with a known human germline immunoglobulin light chain sequence, the 5F4 light chain uses a V _{L segment derived from human germline V κ} L15 and a JK segment derived from human germline JK1. Has been proven. Fig. 12 shows the results of arranging the 5F4 V _L sequence with respect to the germline V _{κ L6 sequence. As a result of further analysis of the 5F4 V L} sequence using the Cabot system for determining the CDR regions, as shown in FIGS. 7B and 12 and SEQ ID NOs: 42, 49 and 56, respectively, the CDR1, CDR2 and CD3 regions of the light chain were derived. I was able to make it.

Example 3: Characterization of binding specificity and binding kinetics of anti-PD-1 human monoclonal antibody

In this Example, the binding affinity and binding kinetics of the anti-PD-1 antibody were observed by Biacore analysis. Binding specificity and cross-competition were observed by flow cytometry.

Binding affinity and kinetics

Anti-PD-1 antibodies were characterized for affinity and binding kinetics through Biacore AB, Uppsala, Sweden. The purified recombinant human PD-1 fusion protein was bound to a CM5 chip (carboxy methyl dextran coated chip) via a primary amine [using standard amine coupling chemistry and kits provided by Biacore]. The binding ability was measured by passing the antibody (concentration = 267 nM) in HBS EP buffer (provided by Biacore AB) at a flow rate of 50 μl/ml. Antigen-antibody binding kinetics were observed for 3 minutes and dissociation kinetics for 7 minutes. The binding and dissociation curves were fitted to a 1:1 Langmuir binding model using BIA evaluation software (Biacore AB). In order to minimize the effect of binding properties in measuring the binding constant, the first section of data corresponding to the binding and dissociation steps was used for correction. _{The K D} , k _on and k _off values measured in this way are shown in Table 2.

Binding specificity by flow cytometry

A Chinese hamster ovary (CHO) cell line expressing recombinant human PD-1 on the cell surface was developed and used to measure the specificity of the PD-1 human monoclonal antibody by flow cytometry. CHO cells were transfected with an expression plasmid containing full-length cDNA encoding dural PD-1. Cells transfected with anti-PD-1 human monoclonal antibody (concentration = 20 µg/ml) were incubated to evaluate the binding ability of 5C4 and 4H1 anti-PD-1 human monoclonal antibodies. The cells were washed and observed for binding to FITC-labeled anti-human IgG Ab. Flow cytometry analysis was performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). The results are shown in Figs. 13A (5C4) and 13B (4H1). The anti-PD-1 human monoclonal antibody bound to CHO cells transfected with PD-1, but not to CHO cells that were not transfected with human PD-1. These data demonstrate the specificity of anti-PD-1 human monoclonal antibodies to PD-1.

Binding specificity for other members of the CD28 group (measured by ELISA)

The binding specificity for PD-1 was observed by comparing the anti-PD-1 antibody to the CD28 group member by standard ELISA using four different CD28 group members.

The fusion proteins of members of the CD28 group, ICOS, CTLA-4 and CD28 (R&D Biosystems) were tested for binding to the anti-PD-1 human monoclonal antibodies 17D8, 2D3, 4H1, 5C4 and 4A11. Standard ELISA methods were performed. Anti-PD-1 human monoclonal antibody was added at a concentration of 20 μg/ml. A goat-anti-human IgG (kappa chain-specific) polyclonal antibody conjugated to horseradish peroxidase (HRP) was used as the secondary antibody. The results are shown in FIG. 14. Anti-PD-1 human monoclonal antibodies 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 each bound to PD-1 with high specificity, but did not bind to other members of the CD28 group.

Example 4: Characterization of anti-PD-1 antibody binding to PD-1 expressed on the surface of human and monkey cells

According to flow cytometry, it was tested whether the anti-PD-1 antibody and cells expressing PD-1 on the cell surface were bound.

Activated human T cells, monkey peripheral blood mononuclear cells (PBMC), and CHO cells transfected with PD-1 were each tested for antibody binding. Human T cells and giant cell PBMCs were activated with an anti-CD3 antibody to express PD-1 on the T cells, and then the human anti-PD-1 monoclonal antibody was bound thereto. Cells transfected with the IgG1 or IgG4 type (different concentrations) of the anti-PD-1 human monoclonal antibody were incubated to evaluate the binding of 5C4 and 4H1 anti-PD-1 human monoclonal antibodies. These cells were washed and evaluated for binding with FITC-labeled anti-human IgG Ab. Flow cytometry analysis was performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). The results are shown in Fig. 15A (activated human T cells), Fig. 15B (giant cell monkey PBMC) and Fig. 15C (PD-1-transfected CHO cells). Anti-PD-1 monoclonal antibodies 5C4 and 4H1 bound to activated human T cells, activated monkey PBMC, and CHO cells transfected with human PD-1, which was determined by the mean fluorescence intensity (MFI) by staining. It was measured by. Through these data, it was demonstrated that the anti-PD-1 HuMAb binds to both human and cytomegalovirus cell surface PD-1.

Example 5: Effect of human anti-PD-1 antibody on cell proliferation and cytokine production in mixed lymphocyte response

The mixed lymphocyte response was used to demonstrate the effect of the PD-1 pathway blocking lymphocyte effector cells. In this assay, T cells were tested for proliferation in the presence or absence of an anti-PD-1 HuMAb antibody, secreting IFN-gamma, and secreting IL-2.

Human T cells were purified from PBMCs using a human CD4+ T cell enrichment column (R&D Systems). Each culture solution contained 10 ⁵ purified T cells and 10 ⁴ allogeneic dendritic cells (total volume = 200 μl). Fab fragment portions of anti-PD-1 monoclonal antibodies 5C4, 4H1, 17D8, 2D3 or 5C4 were added (at different antibody concentrations) to each culture. No antibody or the same reference sample control antibody was used as a negative control. Cells were incubated for 5 days at 37°C. After 5 days, 100 µl of the medium was taken from each culture solution and the amount of cytokine was measured. IFN-gamma and other cytokine levels were measured using the OptEIA ELISA kit (BD Biosciences). Cells ^{were labeled with 3} H-thymidine, cultured for an additional 18 hours, and analyzed for cell proliferation. The results are shown in Figs. 16A (T cell proliferation), Fig. 16B (IFN-γ secretion) and Fig. 16C (IL-2 secretion). Anti-PD-1 human monoclonal antibody promoted T cell proliferation, IFN-gamma secretion and IL-2 secretion in a concentration dependent manner. The 5C4-Fab fragment also promoted T cell proliferation, IFN-gamma secretion and IL-2 secretion in a concentration dependent manner. In contrast, cultures containing the reference sample-type control antibody did not increase T cell proliferation, IFN-gamma secretion, and IL-2 secretion.

Example 6: Blockade of binding of PD-1 and ligand by human anti-PD-1 antibody

Using a flow cytometry assay, it was tested whether anti-PD-1 HuMAb blocks the binding of PD-L1 and PD-L2 to PD-1 expressed on transfected CHO cells.

PD-1 expressing CHO cells were suspended in FACS buffer (PBS containing 4% fetal bovine serum). Various concentrations of anti-PD-1 HuMAb 5C4 and 4H1 were added to the cell suspension, which were incubated at 4° C. for 30 minutes. The unbound antibody was washed off and FITC-labeled PD-L1 fusion protein or FITC-labeled PD-L2 fusion protein was added to the tube, followed by incubation at 4° C. for 30 minutes. Flow cytometry analysis was performed using a FACscan flow cytometer (Becton Dickinson, San Jose, CA). The results are shown in Figs. 17A (blocking of PD-L1) and Fig. 17B (blocking of PD-L2). Anti-PD-1 monoclonal antibodies 5C4 and 4H1 blocked the binding of PD-L1 and PD-L2 to CHO cells transfected with human PD-1 [measured by mean fluorescence intensity (MFI) through staining ]. Through this data, it was demonstrated that anti-PD-1 HuMAb blocks the binding of ligands (both PD-L1 and PD-L2) to PD-1 on the cell surface.

Example 7: Effect of human anti-PD-1 antibody on the release of cytokines in human blood

Anti-PD-1 HuMAb was mixed with fresh human whole blood to determine whether this anti-PD-1 HuMAb alone promotes the release of any cytokines from human blood cells.

500 μl of heparinized-fresh human whole blood was added to each well. To each well, 10 μg or 100 μg of anti-PD-1 HuMAb (4H1 or 5C4; 5C4 was added as a reference sample type for IgG1 or IgG4). Some wells were incubated with anti-CD3 antibody as a positive control, or with human IgG1 or IgG4 antibody as the reference sample type-matching negative control. These cells were incubated at 37° C. for 6 hours or for 24 hours. After centrifuging the cells, plasma was collected, and cytokine IFN-gamma, TNF-alpha, IL-2, IL-4, IL-6, IL- were used by using a bead array assay for cytokine cell counting (BD Biosciences). The concentrations of 10 and IL-12 were measured. Each cytokine concentration (pg/ml) is shown in Table 3a (when incubated for 6 hours) and Table 3b (when incubated for 24 hours) below. As a result, when human anti-PD-1 antibodies 5C4 and 4H1 were treated alone, human blood cells were cytokines IFN-gamma, TNF-alpha, IL-2, IL-4, IL-6, and IL-10. And it can be seen that it did not promote the release of any of IL-12.

[Table 3a]

[Table 3b]

Example 8: Effect of anti-PD-1 antibody on apoptosis of T cells

The effect of anti-PD-1 antibody on the induction of apoptosis of T cells was measured using the Annexin V staining test.

As described in Example 5 above, T cells were cultured in mixed lymphocyte reactions. Anti-PD-1 antibody 5C4 was added to the tube at a concentration of 25 μg/ml. Non-specific antibodies were used as controls. Annexin V and propidium iodide were added to this according to the standard method (BD Sciences). The mixture was incubated for 15 minutes in a dark room at room temperature and then analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). The results are shown in FIG. 18. The anti-PD-1 antibody 5C4 had no effect on the apoptosis of T cells.

Example 9: Effect of anti-PD-1 antibody on cytokine secretion from virus positive donors by virus-promoting PBMC cells

In this example, peripheral blood mononuclear cells (PBMCs) were isolated from a donor positive for CMV and exposed to CMV lysates in the presence or absence of an anti-PD-1 antibody, resulting in cytokine secretion promoted by antigens. The effect was observed.

^{2×10 5} human PMBCs (total volume = 200 μl) derived from a donor positive for CMV were cultured and added to each well along with the lysate of CMV-infected cells. The anti-PD-1 HuMAb 5C4 was added to each well at various concentrations over 4 days. After 4 days, 100 µl of the medium was taken from each culture solution, and the cytokine concentration was measured. IFN-gamma levels were measured using the OptEIA ELISA kit (BD Biosciences). These cells ^{were labeled with 3} H-thymidine and cultured for an additional 18 hours, and then analyzed for proliferation of cells. Cell proliferation was analyzed using Cell Titer-Glo reagent (Promega). The results are shown in FIG. 19. Anti-PD-1 HuMab 5C4 secreted more and more IFN-gamma in a concentration dependent manner. From these results, it was found that anti-PD-1 HuMAb can promote the release of IFN-gamma from PBMC cells (PBMC cells stimulated with antigen in advance) in the memory T cell response.

Example 10: Effect of anti-PD-1 antibody on secondary antibody response to antigen

Mice were immunized again with TI-antigen (DNP-Ficoll) and treated with rat anti-mouse-PD-1 antibody or control antibody to observe the effect of anti-PD-1 antibody on the titer of the antibody.

Female C57BL6 mice were divided into 2 groups (6 per group). One group was treated with control rat IgG, and the other group was treated with rat anti-mouse PD-1 antibody. On day 0, these mice were immunized with 5 μg of DNP-Ficoll (TI-antigen) (in 50 μl of CFA) (intraperitoneal injection). Two days before the start of the experiment, one day before, and at the second day after the experiment, a control rat IgG antibody or rat-mPD-1 antibody (200 μg/mouse) was injected intraperitoneally. After 4 weeks, on day 0, mice were injected intraperitoneally with 5 μg of DNP-Ficoll (in 50 μl IFA). Rat anti-mPD-1 antibody or control antibody (200 μg/mouse) was injected intraperitoneally on Days 0 and 1. The titer of the antibody was measured at the 7th day after immunity triggering by a standard ELISA assay. The results are shown in Table 4 below. In mice treated with the anti-mPD-1 antibody, when TI-antigen was administered, the titers of the same reference sample types for IgM and IgG3 were significantly increased compared to mice treated with the control antibody. Through these results, it was demonstrated that treatment with anti-PD-1 can increase the titer of the antibody in response to TI-antigen.

Secondary reaction of murine animals after treatment with anti-PD-1 antibody Antibodies same reference sample type Control group Rat anti-mouse PD-1 antibody P value IgM 606 1200 0.026 IgG 9 15.55 0.18 IgG1 1.2 1.1 0.83 IgG2b 5.05 9.26 0.18 IgG3 21.9 81.2 0.03 The results shown in this table are the average values of the sample type concentration for the same antibody (㎍/㎖)

Example 11: Treatment of In Vivo Tumor Models Using Anti-PD-1 Antibodies

Mice implanted with cancer tumors were treated with an anti-PD-1 antibody in vivo, and the in vivo effect of the antibody on tumor growth was observed. As a positive control, an anti-CTLA-4 antibody was used, because this antibody was found to inhibit tumor growth in vivo.

In this experiment, the anti-PD-1 antibody used was a chimeric rat anti-mouse-PD-1 antibody generated using well known laboratory techniques. To generate rat anti-mouse PD-1 antibodies, rats were immunized with mouse cells transfected to express recombinant mouse PD-1 fusion protein (R&D Systems Catalog No. 1021-PD), and monoclonal by ELISA assay. The antibody was screened for binding to the mouse PD-1 antigen. Thereafter, the rat anti-PD-1 antibody V site was recombinantly bound to the murine IgG1 constant region using standard molecular biological techniques, and whether it binds to mouse PD-1 by ELISA and FACS was again examined. Screened. The chimeric rat anti-mouse-PD-1 antibody as used herein is referred to as 4H2.

For tumor studies, female AJ mice (6-8 weeks old) (Harlan Laboratories) were randomly selected and divided into 6 groups according to body weight. On day 0, 2×10 ⁶ SA1/N fibrosarcoma cells (dissolved in 200 μl of DMEM medium) were implanted subcutaneously on the flanks of mice. Mice were treated with PBS vehicle or 10 mg/kg of antibody. At the start of the experiment, on the 4th, 8th, and 11th days after the initiation, about 200 μl of PBS (containing an antibody or vehicle) was administered intraperitoneally to the animals. Each group contained 10 animals, which could be divided into: (i) vehicle administration group, (ii) control mouse IgG administration group, (iii) control hamster IgG administration group, (iv) hamster Anti-mouse CTLA-4 antibody administration group, and (v) chimeric anti-PD-1 antibody 4H2 administration group. These mice were observed for tumor growth over about 6 weeks, twice a week. Using an electronic calibrator, the tumor was measured three-dimensionally (height × width × length), and thus the volume of the tumor was calculated. When the tumor volume reached a maximum (1500 mm 3) or when the weight of the mouse decreased by 15%, the mice were euthanized. The results are shown in FIG. 20. The average time until the anti-PD-1 antibody reached the maximum tumor volume was extended [25 to 40 days in the control group]. That is, when the anti-PD-1 antibody was treated, tumor growth was directly inhibited.

Example 12: Generation of chimeric (rat-mouse) anti-PD-1 antibody 4H2

Standard hybridoma production methods [Kohler and Milstein (1975) Nature 256:495; And Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York], a rat monoclonal antibody (rat anti-mPD-1) against mouse PD-1 antibody was prepared. It was generated from rats immunized with the mPD-1-hFc fusion protein. Eight hybridomas were subcloned, antibodies were isolated, and then screened for their ability to inhibit the binding of mouse PD-L2 (mPD-L2) and mPD-1. Identify some anti-mPD-1 antibodies capable of blocking the binding of mPD-L2 and mPD-1 (see, for example, the activity of 4H2 (Figure 41)), and some of the antibodies against the mPD-1-Fc fusion protein The binding affinity of was measured by ELISA (FIG. 42).

Antibody 4H2.B3 was further characterized, which antibody is also referred to herein as “4H2”. CHO cells expressing mouse PD-1 were constructed and incubated with 4H2 anti-mPD-1 antibody (concentration range = 200 μg/ml to 0.012 μg/ml), and the binding affinity of 4H2 and PD-1 Was measured. The binding of the anti-mPD-1 antibody and PD-1 expressing CHO cells was detected by incubation with donkey-anti-rat IgG conjugated to FITC, and this was measured by FACS. _{The EC 50} value (50% effective concentration) of the anti-mPD-1 antibody was about 0.38 μg (Fig. 43), and the K _D value was 4.7×10 ⁻⁹ M. In order to observe the degree of inhibition of binding between PD-L1 and PD-1, the same assay was performed [however, cells were incubated with 0.16 μg of mPD-L1-hFc fusion protein], and then conjugated to FITC. The binding of PD-L1 and PD-1 expressing CHO cells was detected by incubation with the goat-anti-human IgG (Fc specific), and the binding signal was measured by FACS (MFI, mean fluorescence intensity). _{The EC 50} value of the anti-mPD-1 antibody was about 0.72 μg (FIG. 44).

For use in mouse tumor models, 4H2 rat anti-mPD-1 is modified, making the mouse immune system not to neutralize immunotherapeutic antibodies (i.e., better pharmacokinetics of the antibody), reducing Fc receptor interactions. By doing so, antibody-dependent intracellular cytotoxic (ADCC) reactions had to be avoided (ie, blockade by anti-PD-1 could be assessed as the extent to which it was compromised by the ADCC effect). The original rat anti-mPD-1 antibody, 4H2, was determined to be a rat IgG2a same reference sample type. Thus, the Fc-portion of the 4H2 antibody was replaced with an Fc-portion derived from the same reference specimen type of mouse IgG1. Using the above-described assay, it was found that the binding affinity of the rat-mouse chimera 4H2 and mPD-1 is comparable to that of the rat 4H2.B3 anti-mPD-1 antibody (FIG. 45). Similarly, the inhibition of binding of PD-L1 and PD-1 was comparable in the case of both antibodies (FIG. 46 ). Therefore, a rat-mouse chimeric 4H2 anti-mPD-1 antibody was used to observe the therapeutic efficacy of anti-PD-1 in combination with anti-CTLA-4.

Example 13: In vivo effect of combination therapy (anti-CTLA-4 and anti-PD-1 antibodies) on tumor establishment and growth

MC38 colorectal cancer cells ^{(PD-L1 -) [N} . Dr. Restifo (National Cancer Institute, Bethesda, MD); Or Jeffrey Schlom (obtained from National Institutes of Health, Bethesda, MD)] was implanted in C57BL/6 mice [2×10 ⁶ cells/mouse]. On day 0 (i.e., the day of implantation of MC38 cells into mice), one of the following substances was administered intraperitoneally to each of the four groups of 10 mice: (1) mouse IgG (control), (2 ) Anti-CTLA-4 monoclonal antibody 9D9 (mouse anti-mouse CTLA-4, J. Allison, Memorial Sloan-Kettering Cancer Center, New York, NY), (3) anti-PD-1 monoclonal antibody 4H2 (As described in Example 6, rat anti-mouse PD-1 is an antibody modified with a mouse Fc region), or (4) anti-CTLA-4 antibody 9D9 and anti-PD-1 antibody 4H2. Thereafter, on the 3rd, 6th and 10th days, the antibody was further injected and administered. When only one antibody was administered, the amount was 10 mg/kg, and the amount when anti-CTLA-4 antibody and anti-PD-1 antibody were administered together was 5 mg/kg for each antibody [ie, total antibody administration Amount = 10 mg/kg). Using an electronic calibrator, the tumor was measured three-dimensionally (height × width × length), and thus the volume of the tumor was calculated. Mice were euthanized when the tumor volume reached a predetermined volume (maximum tumor volume). The results are shown in Table 5 and Fig. 21.

Proportion of tumor-free mice after anti-PD-1 and/or anti-CTLA-4 treatment process Total number of mice observed Mice with tumor removed (%) mIgG1 10 0 Anti-CTLA-4 10 1(10) Anti-PD-1 10 3(30) Anti-CTLA-4 + anti-PD-1 10 6(60)

The tumor volume of 8 mice belonging to the IgG group reached the maximum volume at the result of about 30 days, and ulcerative tumors occurred in the 2 mice (86066 and 87260) belonging to the IgG group (FIG. 21A). In the case of the group administered with only anti-CTLA-4 antibody, the tumor volume of 7 mice reached the maximum tumor volume after about 60 days, and the tumor volume reached the maximum volume after about 30 days, and 1 mouse (84952 ), an ulcerative tumor occurred, the tumor volume was less than 1500 mm 3 in one mouse (85246), and the tumor disappeared in one mouse (86057) (FIG. 21B). In the case of the group treated with only anti-PD-1 antibody, the tumor volume of 6 mice reached the maximum tumor volume by about 60 days, and ulcerative tumors developed in 1 mouse (86055), and 3 Tumors disappeared in mice (84955, 85239 and 86750) (Fig. 21C). In the case of the group administered with anti-CTLA-4 antibody and anti-PD-1 antibody together, the tumor volume of 4 mice reached the maximum tumor volume by about 40 days, and 6 mice (84596, 85240 , 86056, 86071, 86082 and 86761), the tumor disappeared (Fig. 21D).

22 shows an average tumor volume of about 2955 mm 3 (IgG control group) measured at the time of day 21; It was about 655 mm 3 (CTLA-4 antibody alone treatment group), about 510 mm 3 (PD-1 antibody alone treatment group), and about 280 mm 3 (anti-CTLA-4 antibody and anti-PD-1 antibody co-administration group). To indicate. 23 shows the median tumor volume measured at the time of day 21 is about 2715 mm 3 (IgG group); About 625 mm 3 (CTLA-4 antibody alone treatment group); About 525 mm 3 (PD-1 antibody alone treatment group); And about 10 mm 3 (CTLA-4 antibody and PD-1 antibody co-administered group) (reduced to 0 mm 3 until the lapse of 32 days).

Through this study, in the murine tumor model, treatment with CTLA-4 antibody and PD-1 antibody alone did not significantly affect tumor growth, and CTLA-4 antibody and PD-1 antibody were used together. When treated, it was found that it had a significant effect on tumor growth. When CTLA-4 antibody and PD-1 antibody were treated together (5 mg/kg for each antibody), tumor growth was more affected than when only one of the two antibodies (10 mg/kg or more each) was treated. It should be noted that it had a greater impact.

Example 14: In vivo efficacy of combination therapy (anti-CTLA-4 and anti-PD-1 antibodies) on established tumor growth

^{MC38 colon cancer cells (PD-L1 −} ) were transplanted into C57BL/6 mice (2×10 ⁶ cells/mouse) for a sufficient time (about 6 to 7 days) to allow tumor formation. On the sixth day after transplantation (day -1), the tumor size was measured, and mice were randomly divided into 11 groups based on the average tumor volume (about 250 mm 3 ), followed by antibody therapy. On day 0 (i.e., 1 week after MC38 cell transplantation), mice were treated with (1) mouse IgG (control), (2) anti-CTLA-4 monoclonal antibody 9D9, (3) anti-PD- 1 monoclonal antibody 4H2, or (4) anti-CTLA-4 monoclonal antibody 9D9 and anti-PD-1 antibody monoclonal antibody 4H2 (concentration = 10 mg/kg per mouse) were injected by IP. Antibodies were also injected on the 3rd, 6th, and 10th days. Since the level of endotoxin in the monoclonal antibody composition used was low, it was hardly aggregated. Using an electronic calibrator, the tumor was measured three-dimensionally (height × width × length), and thus the volume of the tumor was calculated. Day 0 after antibody injection (when the tumor volume corresponds to about 125 mm 3 at the start of treatment) and on the 3rd, 6th, 10th, 13th, 17th and 17th days after antibody injection After 20 days, the tumor volume was measured. When the size of the tumor reached a predetermined maximum tumor volume (specific tumor volume = 1500 mm 3, and/or when the mouse body weight decreased by about 15%), mice were euthanized.

In the case of the IgG group consisting of a total of 11 mice, the tumor volume reached the maximum tumor volume after about 17 days (FIG. 24A). In the group treated with anti-CTLA-4 antibody alone, the tumor volume of 7 out of 11 mice reached the maximum tumor volume after about 12 days (FIG. 24B). In the group treated with anti-PD-1 antibody alone, the tumor volume of 4 mice reached the maximum tumor volume after about 13 days, and the tumor disappeared in 2 mice (FIG. 24C). In the group treated with anti-CTLA-4 antibody and anti-PD-1 antibody, the tumor volume of one mouse reached the maximum tumor volume after about 17 days, and the tumor volume of one mouse was about The maximum tumor volume was reached after 45 days, and the tumor disappeared after 45 days in 9 mice (FIG. 24D).

25 shows an average tumor volume of about 1485 mm 3 after 10 days (IgG control group); About 1010 mm 3 (CTLA-4 antibody alone administration group); About 695 mm 3 (PD-1 antibody alone administration group); And it indicates that it is about 80 mm 3 (a group administered with an anti-CTLA-4 antibody and an anti-PD-1 antibody). 26 shows a median tumor volume of about 1365 mm 3 after 10 days (IgG control group); About 1060 mm 3 (anti-CTLA-4 antibody alone administration group); About 480 mm 3 (anti-PD-1 antibody alone administration group); And it indicates that it is about 15 mm 3 (a group administered with an anti-CTLA-4 antibody and an anti-PD-1 antibody together) (the volume decreases to 0 mm 3 until the lapse of 17 days).

In this study, in the murine tumor model, when CTLA-4 antibody and PD-1 antibody were administered together, compared to the case where only one of the two antibodies was administered alone, the tumor growth was significant even when the tumor was already established. It is to prove that it has an effect.

Example 15: Dose titer experiment of combination therapy (anti-CTLA-4 and anti-PD-1 antibody) for established tumor growth

Carried out as described in Example 3, sufficient time MC38 colorectal cancer cells (PD-L1 ^-) during the (approximately 6 to 7) is formed to C57BL / 6 mice were transplanted to the (2 × 10 ⁶ cells / mouse) tumor I made it possible. On the 0th, 3rd, 6th and 10th days, a group of 10 mice was injected IP with the following substances: Group (A) mouse IgG (control, 20 mg/kg), Group (B) anti-PD-1 monoclonal antibody 4H2 (10 mg/kg) and mouse IgG (10 mg/kg), group (C) anti-CTLA-4 monoclonal antibody 9D9 (10 mg/kg) And mouse IgG (10 mg/kg), or group (D) anti-CTLA-4 monoclonal antibody 9D9 (10 mg/kg) and anti-PD-1 antibody monoclonal antibody 4H2 (10 mg/kg), Group (E) anti-CTLA-4 monoclonal antibody 9D9 (3 mg/kg) and anti-PD-1 antibody monoclonal antibody 4H2 (3 mg/kg), or group (F) anti-CTLA-4 monoclonal Rhonal antibody 9D9 (1 mg/kg) and anti-PD-1 antibody monoclonal antibody 4H2 (1 mg/kg). Using an electronic calibrator, the tumor was measured three-dimensionally (height × width × length), and thus the volume of the tumor was calculated. At the start of antibody treatment (i.e., when the tumor volume corresponds to about 90 mm 3 on day 0) and on the 3rd, 6th, 10th, 13th, 17th and 20th days after antibody treatment Tumor volume was measured at one day. When the size of the tumor reached a predetermined maximum tumor volume (specific tumor volume = 1500 mm 3, and/or when the mouse body weight decreased by about 15%), mice were euthanized.

27A shows that a total of 10 control mice reached the maximum tumor volume. Fig. 27B shows that in the 10 mg/kg anti-PD-1 antibody-treated group (group B), the tumor volume of 6 mice reached the maximum tumor volume, and the tumor volume of 4 mice was about 750 mm 3 It shows that the following has been reached. Figure 27c shows that in the case of the 10 mg/kg anti-CTLA-4 antibody-treated group (group C), the tumor volume of 3 mice reached the maximum tumor volume, and the tumor volume of 7 mice was about 1000 mm 3 It shows that it was below. Fig. 27D shows that in the group treated with 10 mg/kg of anti-PD-1 antibody and 10 mg/kg of anti-CTLA-4 antibody together (Group D), the tumor volume of two mice is about 1000 mm 3 or less. And the tumor disappeared in 8 mice. Fig. 27e shows that in the group treated with 3 mg/kg of anti-PD-1 antibody and 3 mg/kg of anti-CTLA-4 antibody together (group E), the tumor volume of one mouse reached the maximum tumor volume. It shows that the tumor volume of 7 mice was less than about 500 mm 3, and the tumor disappeared in 2 mice. Fig. 27F shows that in the group treated with 1 mg/kg of anti-PD-1 antibody and 1 mg/kg of anti-CTLA-4 antibody together (group F), the tumor volume of 4 mice was the maximum tumor volume. Reached, and the tumor volume of 5 mice was less than about 1100 mm 3, and also showed that the tumor disappeared in 1 mouse.

27G and 27H show tumor volumes in mice that were continuously administered in the order of anti-PD-1 antibody → anti-CTLA-4 antibody, and mice administered in the reverse order. The mice in Fig. 27g were first administered 10 mg/kg of anti-CTLA-4 (day 0 and 3), and then 10 mg/kg of anti-PD-1 antibody (day 6 and Day 10). The mice of FIG. 27H were first administered with 10 mg/kg of anti-PD-1 antibody (day 0 and 3), and then 10 mg/kg of anti-CTLA-4 antibody (day 6). And day 10). At day 27, for group G, the tumor volume of 8 mice reached the maximum tumor volume, of which 1 mouse tumor was very small (growth was delayed for a significant period, and growth stopped at the end). , The tumor disappeared in one mouse. At day 27, for group H, the tumor volume of 8 mice reached the maximum tumor volume, and tumor disappeared in 2 mice.

Fig. 28 shows the average tumor volume measured on day 10, about 1250 mm 3 (control IgG group); About 470 mm 3 (Group administered with PD-1 antibody and IgG control group); About 290 mm 3 (Group administered with CTLA-4 antibody and IgG control group (measured at the elapse of day 6)); About 40 mm 3 (group administered with anti-CTLA-4 antibody (10 mg/kg) and anti-PD-1 antibody (10 mg/kg) together); About 165 mm 3 (group administered with anti-CTLA-4 antibody (3 mg/kg) and anti-PD-1 antibody (3 mg/kg) together); And it shows that it was about 400mm3 (the group administered with anti-CTLA-4 antibody (1 mg/kg) and anti-PD-1 antibody (1 mg/kg) together). 29 shows the median tumor volume measured on day 13 was about 1680 mm 3 (control IgG group); About 400 mm 3 (Group administered with PD-1 antibody and IgG control group); About 660 mm 3 (Group administered with CTLA-4 antibody and IgG control group); About 0 mm 3 (group administered with anti-CTLA-4 antibody (10 mg/kg) and anti-PD-1 antibody (10 mg/kg) together); About 90 mm 3 (group administered with anti-CTLA-4 antibody (3 mg/kg) and anti-PD-1 antibody (3 mg/kg) together); And it shows that it was about 650mm3 (a group administered with anti-CTLA-4 antibody (1 mg/kg) and anti-PD-1 antibody (1 mg/kg) together). In administration of anti-PD-1 antibody together with anti-CTLA-4 antibody, the number of tumor-free mice (number per group) on day 27 of the study was 8/10 (10 mg/kg), 2/10. (3 mg/kg) and 1/10 (1 mg/kg) (data not shown).

Throughout this study, in a murine tumor model, treatment with CTLA-4 antibody and PD-1 antibody together shows efficacy in a dose-dependent manner, compared to the case where only one of the two antibodies was administered alone, tumor Even if this was already established, it could be demonstrated that it had a significant effect on tumor growth. In addition, antibodies can be administered sequentially [anti-CTLA-4 antibody → anti-PD-1 antibody, or vice versa], and the combination therapy was still superior in efficacy to antibody alone therapy.

Example 16: In vivo effect of combination therapy (anti-CTLA-4 and anti-PD-1 antibodies) on fibrosarcoma establishment and growth

The day, A / J mouse (2 × 10 ⁶ cells / mouse) in SA1 / N fibrosarcoma cells (PD-L1 ^-) is day 0: a (Leach et al. (1996) Science 271 1734-1736) were implanted subcutaneously . At the 1st, 4th, 7th and 11th days after transplantation, mice were injected IP with the following substances: Group (A) PBS ("vehicle") administered alone; Group (B) mouse IgG (control group, 10 mg/kg per mouse), group (C) anti-PD-1 monoclonal antibody 4H2 (10 mg/kg per mouse), group (D) anti-CTLA- 4 monoclonal antibody 9D9 (10 mg/kg or 0.2 mg/kg per mouse), and group (E) anti-PD-1 monoclonal antibody 4H2 (10 mg/kg per mouse) and anti- Co-administration of CTLA-4 monoclonal antibody 9D9 (0.2 mg/kg per mouse). The study continued for 41 days, and the tumor volume was measured over several days during the study (see Figure 29). The tumor volume was measured in three dimensions (height × width × length) using an electronic calibrator, and thus the volume of the tumor was calculated. When the size of the tumor reached a predetermined maximum tumor volume (1500 mm 3) and/or when an ulcerative tumor occurred, the mice were euthanized.

30A and 30B show that in 19 of 20 control mice (9 out of 10 in group A and 10 out of 10 in group B), the tumor volume reached the maximum tumor volume, or the ulcerative tumor was Occurred. 30C shows that the tumor volume of 6 mice in the 10 mg/kg anti-PD-1 antibody treatment group (group C) reached the maximum tumor volume (the tumor volume of 2 mice was 1500 mm 3 or more, and in 4 mice. Ulcerative tumor), and the tumor disappeared in 4 mice. Figure 30d shows that the tumor volume of 5 mice out of the 10 mg/kg anti-CTLA-4 antibody treatment group (group D) reached the maximum tumor volume (the tumor volume of 2 mice was 1500 mm 3 or more, in 3 mice. Ulcerative tumors occurred), the size of the tumor decreased in 1 mouse (tumor volume = about 70 mm 3 ), and the tumor disappeared in 4 mice. Figure 30e shows that the tumor volume of 10 mice out of the 0.2 mg/kg anti-CTLA-4 antibody-treated group (group E) reached the maximum tumor volume (the tumor volume of 6 mice was 1500 mm 3 or more. , 4 animals developed ulcerative tumors). Figure 30f shows that the tumor volume of two mice in the group treated with 10 mg/kg of anti-PD-1 antibody and 0.2 mg/kg of anti-CTLA-4 antibody (group F) reached the maximum tumor volume ( The tumor volume of one animal is more than 1500 mm3, and ulcerative tumors have occurred in one), showing that the tumor disappeared in 8 mice.

31 and 32 show the mean and median tumor volumes, respectively, and show the mean and median tumor volumes in treated and untreated mice during the course of this study. Tumor growth in the mice treated with these antibodies was suppressed compared to the mice treated with the control antibody mouse IgG, and the results are summarized in Table 6.

In Table 6,

TGI = tumor growth inhibition rate; Median values could be calculated only when the tumor volume of 50% or less of the mice reached the maximum tumor volume.

† Groups were defined in Figure 30.

A = vehicle (PBS); B = mouse IgG; C = anti-PD-1, 10 mg/kg; D = anti-CTLA-4, 10 mg/kg; E = anti-CTLA-4, 0.2 mg/kg; And F = anti-PD-1 (10 mg/kg) and anti-CTLA-4 (0.2 mg/kg).

The data also show that the combination therapy using an anti-PD-1 antibody and an anti-CTLA-4 antibody in combination is substantially more effective than when only one of the two antibodies is administered alone. Indeed, when the anti-CTLA-4 antibody was used in the combination therapy at a dose below the therapeutic dose, this combination therapy was more effective than when treated with only a single antibody. These data also could affect the efficacy of antibody monotherapy in the presence of PD-L1 [i.e., expression of PD-L1 on the tumor could also inhibit anti-tumor T cell responses (Fig. 40 Reference)], suggesting that the presence or absence of PD-L1 on the tumor has little effect on the efficacy of treatment with this antibody.

Example 17: In vivo efficacy and dosage titer of ^PD-L1- combination therapy for fibrosarcoma growth (anti-CTLA-4 and anti-PD-1 antibodies)

On the day 0, A/J mice (2×10 ⁶ cells/mouse) were implanted subcutaneously with SA1/N fibrosarcoma cells (PD-L1 ⁻ ) for a time sufficient to establish tumors (about 7 days). On the 7th, 10th, 13th and 16th days after transplantation, the following substances were injected IP into 10 groups consisting of 8 mice with an average tumor volume of 110 mm 3: Group (A) PBS (“Vehicle”) administered alone; Group (B) mouse IgG (control, 10 mg/kg per mouse), group (C) anti-CTLA-4 monoclonal antibody 9D9 (0.25 mg/kg); Group (D) anti-CTLA-4 monoclonal antibody 9D9 (0.5 mg/kg per mouse); Group (E) anti-CTLA-4 monoclonal antibody 9D9 (5 mg/kg); Group (F) anti-PD-1 monoclonal antibody 4H2 (3 mg/kg per mouse), Group (G) anti-PD-1 monoclonal antibody 4H2 (10 mg/kg per mouse), Group (H) co-administration of anti-PD-1 monoclonal antibody 4H2 (10 mg/kg per mouse) and anti-CTLA-4 monoclonal antibody 9D9 (0.25 mg/kg per mouse), group (I) Co-administration of anti-PD-1 monoclonal antibody 4H2 (10 mg/kg per mouse) and anti-CTLA-4 monoclonal antibody 9D9 (0.5 mg/kg per mouse), and group (J) anti-PD Co-administration of -1 monoclonal antibody 4H2 (3 mg/kg per mouse) and anti-CTLA-4 monoclonal antibody 9D9 (0.5 mg/kg per mouse).

On the 10th, 13th, 16th and 19th days after transplantation, two groups of 6 mice with an average tumor volume of 255 mm 3 were injected IP with the following substances: group (K ) Mouse IgG (control, 10 mg/kg per mouse); And group (L) co-administration of anti-PD-1 monoclonal antibody 4H2 (10 mg/kg per mouse) and anti-CTLA-4 monoclonal antibody 9D9 (1 mg/kg per mouse). The study continued for 51 days, and the volume of the tumor was measured over several days during the study (see Figs. 33-38). The tumor volume was measured in three dimensions (height×width×length) using an electronic calibrator, and the tumor volume was calculated in this way. When the size of the tumor reached a predetermined maximum tumor volume (1500 mm 3) and/or when an ulcerative tumor occurred, the mice were euthanized.

Fig. 33 shows the reaction when the first tumor volume of about 110 mm 3 is treated with an immunostimulating antibody. 33A and 33B show that the tumor volume of a total of 16 mice (groups A and B) reached the maximum tumor volume [of these, 15 had a tumor volume of 1500 mm 3 or more, and 1 was an ulcerative tumor. Is created]. Figures 33c to 33e show that tumor-bearing mice respond to anti-CTLA-4 treatment in a dose-dependent manner [for example, in the case of group C administered 0.25 mg/kg, out of 8 mice, The tumor volume of 7 mice reached the maximum tumor volume, and the tumor volume of 1 mouse was less than 200 mm 3, whereas in the case of group E administered 5 mg/kg, 6 out of 8 mice had The tumor volume reached the maximum tumor volume, and the tumor disappeared in 2 mice]. 33F and 33G show that the mice reacted the same regardless of the dose of the anti-PD-1 antibody [3 mg/kg was administered to group F, and 10 mg/kg was administered to group G]. In contrast, tumor growth was significantly reduced in mice administered with 10 or 3 mg/kg of anti-PD-1 antibody and 0.25 or 0.5 mg/kg of anti-CTLA-4 antibody together (groups H, I and J). . For example, FIG. 33J shows that 2 mice out of the group (Group J) administered with 3 mg/kg of anti-PD-1 antibody and 0.5 mg/kg of anti-CTLA-4 antibody together produced ulcerative tumors. The tumor volume of 2 mice was less than 500 mm 3, showing that the tumor disappeared in 4 mice. The unexpected synergistic effect when the anti-PD-1 antibody and the anti-CTLA-4 enclosure were administered together and the surprising efficacy when the anti-CTLA-4 antibody was administered at a dose less than the therapeutic dose were shown in Fig. 34 (average Tumor volume) and FIG. 35 (median tumor volume).

Fig. 36 shows the response when a mouse with a larger tumor was treated with an immunostimulating antibody, and the tumor volume of the mouse at the initial stage (ie, at the time of the first antibody treatment) was about 250 mm 3. Figure 36a shows that the tumor volume of a total of 6 control mice (group K) reached the maximum tumor volume [the tumor volume of 4 mice was 1500 mm 3 or more, and ulcerative tumors occurred in 2 mice]. 36B shows that in the case of the group treated with 10 mg/kg of anti-PD-1 antibody and 1 mg/kg of anti-CTLA-4 antibody together (group L), ulcerative tumors were formed in one mouse, The tumor volume of 4 mice was more than 1500 mm 3, which shows that the tumor disappeared in 1 mouse. The mean tumor volume and the median tumor volume are shown in FIGS. 37 and 38.

Compared to the control antibody mouse IgGFMF-treated mice, the degree of tumor growth inhibition in mice treated with such an antibody is summarized in Table 7 and FIG. 39 below.

In Table 7,

* TGI = tumor growth inhibition rate; Median values could be calculated only when the tumor volume of 50% or less of the mice reached the maximum tumor volume.

† Groups were defined in Figures 33 and 36.

If the size of the initial tumor is smaller: A = vehicle (PBS); B = mouse IgG (10 mg/kg); C = anti-CTLA-4 (0.25 mg/kg); D = anti-CTLA-4 (0.5 mg/kg); E = anti-CTLA-4 (5 mg/kg); F = anti-PD-1 (3 mg/kg); G = anti-PD-1 (10 mg/kg); H = co-administration of anti-PD-1 (10 mg/kg) and anti-CTLA-4 (0.25 mg/kg); I = co-administration of anti-PD-1 (10 mg/kg) and anti-CTLA-4 (0.5 mg/kg); And J = co-administration of anti-PD-1 (3 mg/kg) and anti-CTLA-4 (0.5 mg/kg).

For larger initial tumor size: K = mouse IgG (10 mg/kg); And L = co-administration of anti-PD-1 (10 mg/kg) and anti-CTLA-4 (0.25 mg/kg).

As a result of synthesizing these data, it was found that the combination therapy using an anti-PD-1 antibody and an anti-CTLA-4 antibody is substantially more efficient than when only one of the two antibodies is treated alone. In addition, the dosage of each antibody can surprisingly be reduced without affecting the synergistic effect of the combined use of the immunostimulating therapeutic antibody as described above. Concomitant therapy is still believed to be effective even when the tumor size becomes smaller (i.e., larger).

Example 18: Immunity against tumors in mice when PD-L1-fibrosarcoma cells were retransplanted after anti-PD-1 antibody treatment

After tumor-free mice were treated with anti-PD-1 antibody when transplanted with tumor cells (ie, after treatment similar to the study on efficacy described in Examples 5 and 6), tumor cells Was transplanted again, and immunity against tumor formation was observed after such treatment. Briefly, when first transplanted, SA1/N fibrosarcoma cells (PD-L1 ⁻ ) were implanted subcutaneously into A/J mice (1×10 ⁶ cells/mouse) (day 0). On the 1st, 4th, 7th, 10th, 14th, 17th and 20th days after transplantation, mouse IgG (control group, 10 mg/kg per mouse) or various Doses of anti-PD-1 monoclonal antibody 4H2 (30, 10, 3, 1 and 0.3 mg/kg per mouse) were injected by IP. Tumor formation and its volume were observed with a precision electron calibrator twice a week until the study was completed. In one group of 8 mice, tumors disappeared after treatment with anti-PD-1 antibody [4 of them were 30 mg/kg, 2 were 3 mg/kg, and 1 was 1 mg/kg, And the remaining one was treated with 0.3mg/kg].

^{1×10 6} SA1/N fibrosarcoma cells/mouse were again subcutaneously implanted into 8 treated A/J mice (mouse without tumor). As a control, 1×10 ⁶ SA1/N fibrosarcoma cells/mouse were again subcutaneously implanted into 9 antibody-untreated mice. Until 62 days after transplantation, tumor formation and its volume were observed with a precision electron calibrator twice a week. The tumor volume of a total of 9 untreated (control) mice reached the maximum tumor volume until 22 days after fibrosarcoma cell transplantation. In contrast, in 8 mice that were re-transplanted with fibrosarcoma cells and did not form tumors, tumors did not form until 62 days after transplantation. Figure 47 shows the mean tumor volume in untreated and retransplanted mice. These results indicate that treatment with an immune-promoting antibody, such as anti-PD-1, provides immunity against further tumor formation in the treated individual, even in the presence of tumor-forming cells. there was.

Example 19: In mice after performing single antibody therapy (anti-PD-1) or parallel antibody therapy (PD-L1 ^-when colon cancer cells are re-implanted, co-use of anti-CTLA-4 and anti-PD-1) Immunity against tumors

When the tumor cells were transplanted, the tumor disappeared and the surviving mice were treated with an anti-PD-1 antibody alone, or an anti-PD-1 antibody and an anti-CTLA-4 antibody together. After treatment similar to the study on the described efficacy], tumor cells were transplanted again, and immunity to tumor formation was observed after such treatment. In short, when the first transplant, MC38 fibrosarcoma cells ^{(PD-L1 -) (2} × 10 6 cells / mouse) of C57BL / 6 mice were subcutaneously transplanted to (2 × 10 ⁶ cells / mouse) mice (day 0 ). On the 0th, 3rd, 6th, and 10th days after transplantation, a group of mice was injected IP with one of the following substances: (1) Mouse IgG (control group, 10 mg/kg per mouse) , (2) anti-PD-1 monoclonal antibody 4H2, or (3) co-administration of anti-PD-1 monoclonal antibody 4H2 and anti-CTLA-4 monoclonal antibody 9D9. As described in Example 15, the growth of the tumor was measured with a precision electron calibrator. One group of 11 mice was treated with anti-PD-1 antibody (a total of 2 treatments) or after treatment with anti-PD-1/anti-CTLA-4 antibody together (a total of 9 treatments). The tumor disappeared.

11 treated C57BL/6 mice (tumor-free mice) ^{were re-implanted with 2×10 7} MC38 colon cancer cells (per mouse) (ie, 10 times greater than the initial dose). As a control, 7 untreated mice ^{were transplanted with 2×10 7} MC38 colon cancer cells (per mouse). During the re-implantation experiment (more than 20 days), tumor formation and volume were measured with a precision electron calibrator. FIG. 48 shows that tumors occurred in a total of 7 untreated (control) mice, and the tumor volume reached the maximum tumor volume until day 18 after colon cancer cell transplantation. In contrast, in the case of a total of 11 mice (mouse with no tumors) re-transplanted with colon cancer cells, tumors did not develop until day 18 after transplantation. Figure 49 shows mean tumor volumes for untreated and retransplanted mice. From these data, it can be seen that, similar to antibody monotherapy, when antibody combination therapy is performed, due to PD-1 and CTLA-4 blockade, permanent immunity against tumor recurrence is imparted.

Example 20: In vivo efficacy of combination therapy (anti-CTLA-4 antibody and anti-PD-1 antibody) on established tumor growth

For a sufficient time (for about 10 days) CT26 colon cancer cells were implanted into BAL B/C mice (2×10 ⁶ cells/mouse) to form tumors. At day 10 after transplantation, the size of the tumor was measured, and mice were randomly divided into 5 groups according to the average tumor volume (about 250 mm 3) for subsequent antibody therapy. Day 0 (i.e., 10 days after transplantation of CT26 cells), to mice (1) mouse IgG (control), (2) anti-CTLA-4 monoclonal antibody 9D9, (3) anti-PD -1 monoclonal antibody 4H2, or (4) anti-CTLA-4 monoclonal antibody 9D9 and anti-PD-1 antibody monoclonal antibody 4H2 (10 mg/kg per mouse) were injected by IP. Antibodies were also injected on the 3rd, 6th, and 10th days. Since the used monoclonal antibody composition contained a small amount of endotoxin, almost no aggregates were formed. Using an electronic calibrator, the tumor was measured three-dimensionally (height × width × length), and thus the volume of the tumor was calculated. Day 0 after antibody injection (when the tumor volume is about 125 mm3 at the start of treatment) and on the 3rd, 6th, 10th, 13th, 17th, and 20th days The tumor volume at the time was measured. When the size of the tumor reached a predetermined maximum tumor volume (specific tumor volume = 1500 mm 3, and/or when the mouse body weight decreased by about 15%), mice were euthanized. The results are shown in FIG. 50. Throughout this study, compared to the case where only one of the above two antibodies was administered alone in a murine tumor model, when the CTLA-4 antibody and PD-1 antibody were treated together, the tumor was already established. It was found that it had a significant impact on growth.

Example 21: Effect of human anti-PD-1 antibody on the function of T regulatory cells

T regulatory cells are lymphocyte cells that suppress the immune response. In this example, it was tested whether T regulatory cells have the ability to inhibit IFN-gamma secretion and proliferation of CD4+CD25- T cells in the presence or absence of an anti-PD-1 human monoclonal antibody.

T regulatory cells were purified from PBMCs using a CD4+CD25+ regulatory T cell isolation kit (Miltenyi Biotec). T regulatory cells were added to a mixed lymphocyte reaction (see above) containing purified CD4+CD25- T cells and allogeneic dendritic cells (CD4+CD25- T cells:T regulatory cells ratio = 2:1). Anti-PD-1 monoclonal antibody 5C4 was added at a concentration of 10 μg/ml. One to which no antibody was added or the one to which the same reference sample type antibody was added was used as a negative control. At the lapse of day 5, the culture supernatant was collected, and the amount of cytokines was measured using a Beadlyte cytokine detection system (Upstate). These cells ^{were labeled with 3} H-thymidine, and cultured for an additional 18 hours, and analyzed for cell proliferation. The results are shown in Figs. 51a (T cell proliferation) and 51b (IFN-gamma secretion). When the anti-PD-1 human monoclonal antibody 5C4 was added, proliferation was partially inhibited by T regulatory cells, and the secretion of IFN-gamma by CD4+CD25- T cells was also inhibited, which is called anti-PD-1. This suggests that the antibody affects T regulatory cells.

Example 22: Effect of human anti-PD-1 antibody on T cell activation

In this example, the blocking effect of the PD-1 pathway by anti-PD-1 antibody 5C4 was observed in T cell activation. Purified human CD4+ T cells (Dynal CD4 T cell purification kit) were activated with 1 μg/ml of soluble anti-CD3 antibody (BD) in the presence of autologous monocytes or monocyte-derived dendritic cells (DC). Monocytes were purified using a Miltenyi CD14 monocyte purification kit, and DCs were generated by culturing GM-CSF and IL-4 (PeproTech) and monocytes for 7 days. After activation for 3 days in the presence or absence of titrated anti-PD-1 antibody or irrelevant reference sample control mAb, culture supernatant was collected and ELISA analysis was performed for IFN-γ secretion, and further assay In the final 18 hours, titrated thymidine was added to measure T cell proliferation. The results are shown in FIGS. 52A and 52B, which proved that as a result of blocking PD-1 by an anti-PD-1 antibody, T cell proliferation and IFN-γ secretion increased. In T cell activation (especially IFN-γ secretion), a synergistic effect of anti-PD-1 antibody and anti-CTLA-4 antibody in the presence of monocytes was also observed.

Example 23: Evaluation of ADCC effect of anti-PD-1 antibody

In this example, an antibody-dependent intracellular cytotoxicity (ADCC) assay was performed to evaluate whether an anti-PD-1 antibody could induce ADCC into target cells. Two types of 5C4 (one with the Fc region of human IgG1 (5C4-IgG1) and the other with the Fc region of human IgG4 (5C4-IgG4) in this assay) were tested. In this assay, Delfia Cell Cytotoxicity Kit (Perkin Elmer) was used. Briefly, human CD4 T cells (Dynal CD4 T cell purification kit) purified with plate-bound anti-CD3 antibody (BD) were activated to induce the expression of PD-1. Thereafter, the target activated with CD4 T cells was labeled with a BATDA reagent. Labeled CD4 T cells were added to a V-bottom 96-well plate, and then human PBMC (effector: target cell ratio (E/T) = 50:1) was added thereto to design an antibody. After incubation at 37° C. for 1 hour, the plate was centrifuged. After the supernatant was transferred to a flat bottom 96-well plate, the plate was read with a RubyStar plate reader. From the results, it was found that 5C4-IgG4 did not mediate ADCC on activated CD4 T cells, whereas 5C4-IgG1 mediated ADCC on activated CD4 T cells (Fig. 53), which is, ADCC activity. It was suggested that it is related to the Fc region of this anti-PD-1 antibody.

Example 24: Evaluation of complement-dependent cytotoxicity of anti-PD-1 antibody

In this example, complement dependent cytotoxicity (CDC) of the anti-PD-1 antibody was observed. In this assay, two forms of 5C4 (one having the Fc region of human IgG1 (5C4-IgG1) and the other having the Fc region of human IgG4 (5C4-IgG4)) were tested. Briefly, purified human CD4 T cells (Dynal CD4 T cell purification kit) were activated with a plate-bound anti-CD3 antibody (BD) to induce the expression of PD-1. Anti-PD-1 antibody (5C4) and control antibody were serially diluted (50 μg/ml → 640 pg/ml) and tested for CDC in the presence of human complement (Quidel-A113). Cytotoxicity was measured using Alamar blue (Biosource International). This plate was read on a fluorescent plate reader (EX530 EM590). The number of viable cells was proportional to the unit of fluorescence. From the results, it was found that neither 5C4-IgG1 nor 5C4-IgG4 mediated CDC on activated CD4 T cells, whereas positive control antibody (anti-HLA-ABC antibody) mediated (FIG. 54 ). .

Example 25: Evaluation of PD-1 expression in human T cells

In this example, human PBMCs derived from different donors by FACS were observed for PD-1 expression in various cell subsets. In this assay, a biotinylated anti-PD-1 antibody (i.e., an antibody having a higher sensitivity than a commercially available anti-PD-1 antibody in the detection of PD-1 molecules on the cell surface) was used. Binding antibodies were detected using PE-conjugated streptavidin. Flow cytometry analysis was performed using a FACScan flow cytometer (Becton Dickinson) and Flowjo software (Tree Star). PD-1 expression was detected in some peripheral human T cells, but not B cells or monocytes. Further observations of the T cell subset revealed that PD-1 was expressed in CD4 and CD8 memory and effector T cells, but not in untreated CD4 or CD8 T cells.

The scope of the invention is not limited by the specific embodiments disclosed herein. In fact, in addition to those disclosed herein, various modifications may be made to the present invention, as will be well known to those skilled in the art through the above detailed description and the accompanying drawings. Such modifications are also included within the scope of the appended claims. Therefore, the present invention is limited only by the matters of the appended claims, along with both the matters defined by the claims and the matters falling within the equivalent range.

SEQUENCE LISTING <110> Medarex, Inc. <110> Ono Pharmaceutical Co., LTD. <120> HUMAN MONOCLONAL ANTIBODIES TO PROGRAMMED DEATH 1 (PD-1) AND METHODS FOR TREATING CANCER USING ANTI-PD-1 ANTIBODIES ALONE OR IN COMBINATION WITH OTHER IMMUNOTHERAPEUTICS <130> GEOP-50 <150> US 60/679,466 <151> 2005-05-09 <150> US 60/738,434 <151> 2005-11-21 <150> US 60/748,919 <151> 2005-12-08 <160> 74 <170> PatentIn version 3.1 <210> 1 <211> 113 <212> PRT <213> Homo sapiens <400> 1 Gln Val Gln Leu Val Glu Ser Gly Gly Asp Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ala Phe Ser Asn Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Met Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <210> 2 <211> 113 <212> PRT <213> Homo sapiens <400> 2 Gln Val Gln Leu Val Glu Ser Gly Gly Asp Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Thr Asn Tyr 20 25 30 Gly Phe His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <210> 3 <211> 113 <212> PRT <213> Homo sapiens <400> 3 Gln Val Tyr Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Asn Val Asp His Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <210> 4 <211> 113 <212> PRT <213> Homo sapiens <400> 4 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <210> 5 <211> 121 <212> PRT <213> Homo sapiens <400> 5 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Ser Arg Ser 20 25 30 Ser Phe Phe Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Val Arg Asp Tyr Asp Ile Leu Thr Gly Asp Glu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 6 <211> 113 <212> PRT <213> Homo sapiens <400> 6 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Thr Ser Gly Ile Thr Phe Ser Ser Tyr 20 25 30 Gly Phe His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Leu Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Thr Gly Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <210> 7 <211> 121 <212> PRT <213> Homo sapiens <400> 7 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Ser Gly Gly Ser Leu Ser Arg Ser 20 25 30 Ser Tyr Phe Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Ala Ser Ile Phe Tyr Ser Gly Glu Thr Tyr Phe Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Arg Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Tyr Asp Ile Leu Thr Gly Asp Glu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 8 <211> 107 <212> PRT <213> Homo sapiens <400> 8 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Ile Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 9 <211> 107 <212> PRT <213> Homo sapiens <400> 9 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Thr Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 10 <211> 107 <212> PRT <213> Homo sapiens <400> 10 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 11 <211> 107 <212> PRT <213> Homo sapiens <400> 11 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 12 <211> 107 <212> PRT <213> Homo sapiens <400> 12 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Asn Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 13 <211> 107 <212> PRT <213> Homo sapiens <400> 13 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 14 <211> 107 <212> PRT <213> Homo sapiens <400> 14 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 15 <211> 5 <212> PRT <213> Homo sapiens <400> 15 Asn Tyr Gly Met His 1 5 <210> 16 <211> 5 <212> PRT <213> Homo sapiens <400> 16 Asn Tyr Gly Phe His 1 5 <210> 17 <211> 5 <212> PRT <213> Homo sapiens <400> 17 Asn Tyr Gly Met His 1 5 <210> 18 <211> 5 <212> PRT <213> Homo sapiens <400> 18 Asn Ser Gly Met His 1 5 <210> 19 <211> 7 <212> PRT <213> Homo sapiens <400> 19 Arg Ser Ser Phe Phe Trp Gly 1 5 <210> 20 <211> 5 <212> PRT <213> Homo sapiens <400> 20 Ser Tyr Gly Phe His 1 5 <210> 21 <211> 7 <212> PRT <213> Homo sapiens <400> 21 Arg Ser Ser Tyr Phe Trp Gly 1 5 <210> 22 <211> 17 <212> PRT <213> Homo sapiens <400> 22 Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 23 <211> 17 <212> PRT <213> Homo sapiens <400> 23 Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 24 <211> 17 <212> PRT <213> Homo sapiens <400> 24 Leu Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 25 <211> 17 <212> PRT <213> Homo sapiens <400> 25 Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 26 <211> 16 <212> PRT <213> Homo sapiens <400> 26 Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 27 <211> 17 <212> PRT <213> Homo sapiens <400> 27 Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 28 <211> 16 <212> PRT <213> Homo sapiens <400> 28 Ser Ile Phe Tyr Ser Gly Glu Thr Tyr Phe Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 29 <211> 4 <212> PRT <213> Homo sapiens <400> 29 Asn Asp Asp Tyr One <210> 30 <211> 4 <212> PRT <213> Homo sapiens <400> 30 Gly Asp Asp Tyr One <210> 31 <211> 4 <212> PRT <213> Homo sapiens <400> 31 Asn Val Asp His One <210> 32 <211> 4 <212> PRT <213> Homo sapiens <400> 32 Asn Asp Asp Tyr One <210> 33 <211> 11 <212> PRT <213> Homo sapiens <400> 33 Asp Tyr Asp Ile Leu Thr Gly Asp Glu Asp Tyr 1 5 10 <210> 34 <211> 4 <212> PRT <213> Homo sapiens <400> 34 Gly Asp Asp Tyr One <210> 35 <211> 11 <212> PRT <213> Homo sapiens <400> 35 Asp Tyr Asp Ile Leu Thr Gly Asp Glu Asp Tyr 1 5 10 <210> 36 <211> 11 <212> PRT <213> Homo sapiens <400> 36 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 37 <211> 11 <212> PRT <213> Homo sapiens <400> 37 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 38 <211> 11 <212> PRT <213> Homo sapiens <400> 38 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 39 <211> 11 <212> PRT <213> Homo sapiens <400> 39 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 40 <211> 11 <212> PRT <213> Homo sapiens <400> 40 Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala 1 5 10 <210> 41 <211> 11 <212> PRT <213> Homo sapiens <400> 41 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 42 <211> 11 <212> PRT <213> Homo sapiens <400> 42 Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala 1 5 10 <210> 43 <211> 7 <212> PRT <213> Homo sapiens <400> 43 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 44 <211> 7 <212> PRT <213> Homo sapiens <400> 44 Asp Thr Ser Asn Arg Ala Thr 1 5 <210> 45 <211> 7 <212> PRT <213> Homo sapiens <400> 45 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 46 <211> 7 <212> PRT <213> Homo sapiens <400> 46 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 47 <211> 7 <212> PRT <213> Homo sapiens <400> 47 Ala Ala Ser Asn Leu Arg Ser 1 5 <210> 48 <211> 7 <212> PRT <213> Homo sapiens <400> 48 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 49 <211> 7 <212> PRT <213> Homo sapiens <400> 49 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 50 <211> 9 <212> PRT <213> Homo sapiens <400> 50 Gln Gln Arg Ser Asn Trp Pro Leu Thr 1 5 <210> 51 <211> 9 <212> PRT <213> Homo sapiens <400> 51 Gln Gln Arg Ser Asn Trp Pro Leu Thr 1 5 <210> 52 <211> 9 <212> PRT <213> Homo sapiens <400> 52 Gln Gln Ser Ser Asn Trp Pro Arg Thr 1 5 <210> 53 <211> 9 <212> PRT <213> Homo sapiens <400> 53 Gln Gln Ser Ser Asn Trp Pro Arg Thr 1 5 <210> 54 <211> 9 <212> PRT <213> Homo sapiens <400> 54 Gln Gln Tyr Tyr Ser Tyr Pro Arg Thr 1 5 <210> 55 <211> 9 <212> PRT <213> Homo sapiens <400> 55 Gln Gln Arg Ser Asn Trp Pro Leu Thr 1 5 <210> 56 <211> 9 <212> PRT <213> Homo sapiens <400> 56 Gln Gln Tyr Tyr Ser Tyr Pro Arg Thr 1 5 <210> 57 <211> 339 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(339) <400> 57 cag gtg cag ctg gtg gag tct ggg gga gac gtg gtc cag cct ggg ggg 48 Gln Val Gln Leu Val Glu Ser Gly Gly Asp Val Val Gln Pro Gly Gly 1 5 10 15 tcc ctg aga ctc tcc tgt gca gcg tct gga gtc gcc ttc agt aac tat 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ala Phe Ser Asn Tyr 20 25 30 ggc atg cac tgg gtc cgc cag gct ccc ggc aag ggg ctg gag tgg gtg 144 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 gca gtt atc tgg tat gat gga agt aat aaa tac tat gca gac tcc gtg 192 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 aag ggc cgg ttc acc atc tcc aga gac aat tcc aag aac atg ctc tat 240 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Met Leu Tyr 65 70 75 80 ctg caa atg aac agc ctg aga gcc gag gac acg gct atg tat tac tgt 288 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 gcg agg aac gat gac tac tgg ggc cag gga acc ctg gtc acc gtc tcc 336 Ala Arg Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 tca 339 Ser <210> 58 <211> 339 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(339) <400> 58 cag gtg cag ctg gtg gaa tct ggg gga gac gtg gtc cag cct ggg agg 48 Gln Val Gln Leu Val Glu Ser Gly Gly Asp Val Val Gln Pro Gly Arg 1 5 10 15 tcc ctg aga ctc tcc tgt gca gcg tct gga tta acc ttc act aac tat 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Thr Asn Tyr 20 25 30 ggc ttc cac tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg 144 Gly Phe His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 gct gtt ata tgg tat gat gga agt aag aaa tat tat gca gac tcc gtg 192 Ala Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val 50 55 60 aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat 240 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 ctg caa atg aac aac ctg aga gcc gag gac acg gct gtg tat tac tgt 288 Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcg act ggg gat gac tac tgg ggc cag gga acc ctg gtc acc gtc tcc 336 Ala Thr Gly Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 tca 339 Ser <210> 59 <211> 339 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(339) <400> 59 cag gtg tac ttg gta gag tct ggg gga ggc gtg gtc cag cct ggg agg 48 Gln Val Tyr Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 tcc ctg aga ctc tcc tgt gca gcg tct gga ttc acc ttc agt aac tat 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg 144 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 gca ctt ata tgg tat gat gga agt aat aaa tac tat gca gac tcc gtg 192 Ala Leu Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat 240 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 ctg caa atg acc agt ctg aga gtc gag gac acg gct gtg tat tat tgt 288 Leu Gln Met Thr Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcg agc aac gtt gac cat tgg ggc cag gga acc ctg gtc acc gtc tcc 336 Ala Ser Asn Val Asp His Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 tca 339 Ser <210> 60 <211> 339 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(339) <400> 60 cag gtg cag ctg gtg gag tct ggg gga ggc gtg gtc cag cct ggg agg 48 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 tcc ctg aga ctc gac tgt aaa gcg tct gga atc acc ttc agt aac tct 96 Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg 144 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 gca gtt att tgg tat gat gga agt aaa aga tac tat gca gac tcc gtg 192 Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg ttt 240 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 ctg caa atg aac agc ctg aga gcc gag gac acg gct gtg tat tac tgt 288 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcg aca aac gac gac tac tgg ggc cag gga acc ctg gtc acc gtc tcc 336 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 tca 339 Ser <210> 61 <211> 363 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(363) <400> 61 cag ctg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gag 48 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 acc ctg tcc ctc acc tgc act gtc tct ggt ggc tcc ctc agc agg agt 96 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Ser Arg Ser 20 25 30 agt ttc ttc tgg ggc tgg atc cgt cag ccc cca ggg aag gga ctg gag 144 Ser Phe Phe Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 tgg att ggg agt atc tat tat agt ggg agc acc tac tac aac ccg tcc 192 Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 ctc aag agt cga gtc acc ata tcc gta gac acg tcc aag aac cag ttc 240 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 tcc ctg aag ctg agc tct gtg acc gcc gca gac acg gct gtg tat tac 288 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 tgt gtg aga gat tac gat att ttg act ggc gac gag gac tac tgg ggc 336 Cys Val Arg Asp Tyr Asp Ile Leu Thr Gly Asp Glu Asp Tyr Trp Gly 100 105 110 cag gga acc ctg gtc acc gtg tcc tca 363 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 62 <211> 339 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(339) <400> 62 cag gtg cag ctg gtg gag tct ggg gga ggc gtg gtc cag cct ggg agg 48 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 tcc ctg aga ctc tcc tgt aca acg tct gga atc acc ttc agt agc tat 96 Ser Leu Arg Leu Ser Cys Thr Thr Ser Gly Ile Thr Phe Ser Ser Tyr 20 25 30 ggc ttt cac tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg 144 Gly Phe His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 gca gtg ata tgg tat gat gga agt aaa aaa tac tat gca gac tcc gtg 192 Ala Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val 50 55 60 aag ggc cga ttc acc ctc tcc aga gac gat tcc aag aac acg ctg tat 240 Lys Gly Arg Phe Thr Leu Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr 65 70 75 80 ctg caa atg aac agc ctg aga gcc gag gac acg gct gtg tat tac tgt 288 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gtt act ggg gat gac tac tgg ggc cag gga acc ctg gtc acc gtc tcc 336 Val Thr Gly Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 tca 339 Ser <210> 63 <211> 363 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(363) <400> 63 cag ctg cag ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gag 48 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 acc ctg tcc ctc acc tgc tct gtc tct ggt ggc tcc ctc agc agg agt 96 Thr Leu Ser Leu Thr Cys Ser Val Ser Gly Gly Ser Leu Ser Arg Ser 20 25 30 agt tac ttc tgg ggc tgg atc cgc cag ccc cca ggg aag ggg ctg gag 144 Ser Tyr Phe Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 tgg att gcg agt atc ttt tat agt ggg gaa acc tac ttc aat ccg tcc 192 Trp Ile Ala Ser Ile Phe Tyr Ser Gly Glu Thr Tyr Phe Asn Pro Ser 50 55 60 ctc aag agt cga gtc acc ata tcc gta gac acg tcc agg aac cag ttc 240 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Arg Asn Gln Phe 65 70 75 80 tcc ctg aag ctg agc tct gtg acc gcc gca gac acg gct gtg tat tac 288 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 tgt gcg aga gat tac gat att ttg act ggc gac gag gac tac tgg ggc 336 Cys Ala Arg Asp Tyr Asp Ile Leu Thr Gly Asp Glu Asp Tyr Trp Gly 100 105 110 cag gga acc ctg gtc acc gtc tcc tca 363 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 64 <211> 321 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(321) <400> 64 gaa att gtg ttg aca cag tct cca gcc acc ctg tct ttg tct cca ggg 48 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agc agc tac 96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 tta gcc tgg tac caa cag aaa cct ggc cag gct ccc agg ctc atc atc 144 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Ile Ile 35 40 45 tat gat gca tcc aac agg gcc act ggc atc cca gcc agg ttc agt ggc 192 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 agt ggg tct ggg aca gac ttc act ctc acc atc agc agc cta gag cct 240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 gaa gat ttt gca gtt tat tac tgt cag cag cgt agc aac tgg cct ctc 288 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu 85 90 95 act ttc ggc gga ggg acc aag gtg gag atc aaa 321 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 65 <211> 321 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(321) <400> 65 gaa att gtg ttg aca cag tct cca gcc acc ctg tct ttg tct cca ggg 48 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 gaa aga gcc acc ctg tcc tgc agg gcc agt cag agt gtt agc agc tac 96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 tta gcc tgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc atc 144 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 tat gat aca tcc aac agg gcc act ggc atc cca gcc agg ttc agt ggc 192 Tyr Asp Thr Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 agt ggg tct ggg aca gac ttc act ctc acc atc agc agc cta gag cct 240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 gaa gat ttt gca gtt tat tac tgt cag cag cgt agc aac tgg ccg ctc 288 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu 85 90 95 act ttc ggc gga ggg acc aag gtg gag atc aaa 321 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 66 <211> 324 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(321) <400> 66 gaa att gtg ttg aca cag tct cca gcc acc ctg tct ttg tct cca ggg 48 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agt agt tac 96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 tta gcc tgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc atc 144 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 tat gat gca tcc aac agg gcc act ggc atc cca gcc agg ttc agt ggc 192 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 agt ggg tct ggg aca gac ttc act ctc acc atc agc agc cta gag cct 240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 gaa gat ttt gca gtt tat tac tgt cag cag agt agc aac tgg cct cgg 288 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 90 95 acg ttc ggc caa ggg acc aag gtg gaa atc aaa 321 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 67 <211> 321 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(321) <400> 67 gaa att gtg ttg aca cag tct cca gcc acc ctg tct ttg tct cca ggg 48 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agt agt tac 96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 tta gcc tgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc atc 144 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 tat gat gca tcc aac agg gcc act ggc atc cca gcc agg ttc agt ggc 192 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 agt ggg tct ggg aca gac ttc act ctc acc atc agc agc cta gag cct 240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 gaa gat ttt gca gtt tat tac tgt cag cag agt agc aac tgg cct cgg 288 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 90 95 acg ttc ggc caa ggg acc aag gtg gaa atc aaa 321 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 68 <211> 321 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(321) <400> 68 gac atc cag atg acc cag tct cca tcc tca ctg tct gca tct gtg gga 48 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 gac aga gtc tcc atc act tgt cgg gcg agt cag ggt att agc agc tgg 96 Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 tta gcc tgg tat cag cag aaa cca gag aaa gcc cct aag tcc ctg atc 144 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 tat gct gca tcc aat tta cga agt ggg gtc cca tca agg ttc agc ggc 192 Tyr Ala Ala Ser Asn Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 agt gga tct ggg aca gat ttc act ctc acc atc agc agc ctg cag cct 240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 gaa gat ttt gca act tat tac tgc caa cag tat tat agt tac cct agg 288 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Arg 85 90 95 acg ttc ggc caa ggg acc aag gtg gaa atc aaa 321 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 69 <211> 321 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(321) <400> 69 gaa att gtg ttg aca cag tct cca gcc acc ctg tct ttg tct cca ggg 48 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agc agc tac 96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 tta gcc tgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc atc 144 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 tat gat gca tcc aac agg gcc act ggc atc cca gcc agg ttc agt ggc 192 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 agt ggg tct ggg aca gac ttc act ctc acc atc agc agc cta gag cct 240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 gaa gat ttt gca gtt tat tac tgt cag cag cgt agc aac tgg cct ctc 288 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu 85 90 95 act ttc ggc gga ggg acc aag gtg gag atc aaa 321 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 70 <211> 321 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(321) <400> 70 gac atc cag atg acc cag tct cca tcc tca ctg tct gca tct gta gga 48 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 gac aga gtc acc atc act tgt cgg gcg agt cag ggt att agc agc tgg 96 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 tta gcc tgg tat cag cag aaa cca gag aaa gcc cct aag tcc ctg atc 144 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 tat gct gca tcc agt ttg caa agt ggg gtc cca tca agg ttc agc ggc 192 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 agt gga tct ggg aca gat ttc act ctc acc atc agc agc ctg cag cct 240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 gaa gat ttt gca act tat tac tgc caa cag tat tat agt tac cct agg 288 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Arg 85 90 95 acg ttc ggc caa ggg acc aag gtg gaa atc aaa 321 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 71 <211> 98 <212> PRT <213> Homo sapiens <400> 71 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> 72 <211> 95 <212> PRT <213> Homo sapiens <400> 72 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Ile Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro 85 90 95 <210> 73 <211> 99 <212> PRT <213> Homo sapiens <400> 73 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn His Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg <210> 74 <211> 95 <212> PRT <213> Homo sapiens <400> 74 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro 85 90 95

Claims (21)

(a) a heavy chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 18;
A heavy chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 25;
A heavy chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 32;
Light chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 39;
Light chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 46; And
Light chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 53;
(b) a heavy chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 15;
A heavy chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 22;
A heavy chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 29;
Light chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 36;
Light chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 43; And
Light chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 50;
(c) a heavy chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 16;
A heavy chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 23;
A heavy chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 30;
Light chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 37;
Light chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 44; And
Light chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 51;
(d) a heavy chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 17;
A heavy chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 24;
A heavy chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 31;
Light chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 38;
Light chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 45; And
Light chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 52; or
(e) a heavy chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 20;
A heavy chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 27;
A heavy chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 34;
Light chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 41;
Light chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 48; And
Light chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 55;
A pharmaceutical composition for treating cancer comprising a monoclonal antibody or an antigen-binding portion thereof, the monoclonal antibody specifically binding to human PD-1. The method of claim 1,
The antibody
(a) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 4; And a light chain variable portion having the amino acid sequence set forth in SEQ ID NO: 11;
(b) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1; And a light chain variable portion having the amino acid sequence set forth in SEQ ID NO: 8;
(c) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 2; And a light chain variable portion having the amino acid sequence set forth in SEQ ID NO: 9;
(d) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 3; And a light chain variable portion having the amino acid sequence set forth in SEQ ID NO: 10; or
(e) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 6; And a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 13. (a) binds to PD-1 with a KD value of 1 × 10 ⁻⁸ M or less as determined by BIAcore analysis; (b) a pharmaceutical composition for treating cancer comprising a monoclonal antibody or antigen-binding portion thereof, which competes with a reference antibody for binding to human PD-1,
Wherein said reference antibody is a human monoclonal antibody that specifically binds to human PD-1 and comprises a heavy chain variable region having an amino acid sequence as set out in SEQ ID NO: 4 and a light chain variable region having an amino acid sequence as set out in SEQ ID NO: 11 Composition. 4. The method according to any one of claims 1 to 3,
Wherein said monoclonal antibody is an IgG1 or IgG4 isotype. 5. The method of claim 4,
Wherein said monoclonal antibody is an IgG4 isotypic sample. 4. The method according to any one of claims 1 to 3,
The monoclonal antibody is a chimeric, humanized or human antibody or part thereof. The method according to claim 6,
The monoclonal antibody is a human monoclonal antibody or part thereof, pharmaceutical composition. IgG4 is a reference specimen that specifically binds to native human PD-1 expressed on the cell surface,
A heavy chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 18;
A heavy chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 25;
A heavy chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 32;
Light chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 39;
Light chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 46; And
Light chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 53;
Human monoclonal antibody or antigen-binding portion thereof comprising a pharmaceutical composition for treating cancer. 9. The method of claim 8,
Pharmaceutical composition, wherein the human monoclonal antibody is a human monoclonal antibody comprising a heavy chain variable region having an amino acid sequence as set out in SEQ ID NO: 4 and a light chain variable region having an amino acid sequence as set out in SEQ ID NO: 11 The method according to any one of claims 1 to 3, 8, and 9,
The cancer is a pharmaceutical composition that is selected from the group consisting of melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer and lung cancer. The method of claim 10,
The lung cancer is a non-small cell lung cancer pharmaceutical composition. (a) a heavy chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 18;
A heavy chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 25;
A heavy chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 32;
Light chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 39;
Light chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 46; And
Light chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 53;
(b) a heavy chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 15;
A heavy chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 22;
A heavy chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 29;
Light chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 36;
Light chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 43; And
Light chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 50;
(c) a heavy chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 16;
A heavy chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 23;
A heavy chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 30;
Light chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 37;
Light chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 44; And
Light chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 51;
(d) a heavy chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 17;
A heavy chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 24;
A heavy chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 31;
Light chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 38;
Light chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 45; And
Light chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 52; or
(e) a heavy chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 20;
A heavy chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 27;
A heavy chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 34;
Light chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 41;
Light chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 48; And
Light chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 55;
A pharmaceutical composition for treating an infectious disease comprising a monoclonal antibody or an antigen-binding portion thereof, the monoclonal antibody specifically binding to human PD-1. The method of claim 12,
The antibody
(a) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 4; And a light chain variable portion having the amino acid sequence set forth in SEQ ID NO: 11;
(b) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1; And a light chain variable portion having the amino acid sequence set forth in SEQ ID NO: 8;
(c) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 2; And a light chain variable portion having the amino acid sequence set forth in SEQ ID NO: 9;
(d) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 3; And a light chain variable portion having the amino acid sequence set forth in SEQ ID NO: 10; or
(e) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 6; And a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 13. (a) binds to PD-1 with a KD value of 1 × 10 ⁻⁸ M or less as determined by BIAcore analysis; (b) a pharmaceutical composition for treating an infectious disease comprising a monoclonal antibody or antigen-binding portion thereof, which competes with a reference antibody for binding to human PD-1,
The reference antibody is a human monoclonal antibody that specifically binds to human PD-1 and comprises a heavy chain variable region having an amino acid sequence as set out in SEQ ID NO: 4 and a light chain variable region having an amino acid sequence as set out in SEQ ID NO: 11. Pharmaceutical compositions for the treatment of diseases. 15. The method according to any one of claims 12 to 14,
Wherein said monoclonal antibody is an IgG1 or IgG4 isotype. 16. The method of claim 15,
Wherein said monoclonal antibody is an IgG4 isotypic sample. 15. The method according to any one of claims 12 to 14,
The monoclonal antibody is a chimeric, humanized or human antibody or part thereof. 18. The method of claim 17,
The monoclonal antibody is a human monoclonal antibody or part thereof, pharmaceutical composition. IgG4 is a reference specimen that specifically binds to native human PD-1 expressed on the cell surface,
A heavy chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 18;
A heavy chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 25;
A heavy chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 32;
Light chain variable region CDR1 having the amino acid sequence set forth in SEQ ID NO: 39;
Light chain variable region CDR2 having the amino acid sequence set forth in SEQ ID NO: 46; And
Light chain variable region CDR3 having the amino acid sequence set forth in SEQ ID NO: 53;
Pharmaceutical composition for the treatment of infectious diseases comprising a human monoclonal antibody or antigen-binding portion thereof. 20. The method of claim 19,
Pharmaceutical composition, wherein the human monoclonal antibody is a human monoclonal antibody comprising a heavy chain variable region having an amino acid sequence as set out in SEQ ID NO: 4 and a light chain variable region having an amino acid sequence as set out in SEQ ID NO: 11 The method according to any one of claims 12 to 14, 19 and 20,
Infectious diseases,
HIV, hepatitis virus, herpes virus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackie virus, cornovirus (cornovirus), respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus (vaccinia) virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus A pharmaceutical composition which is a pathogenic infection with a virus selected from the group consisting of.

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